Rapidly degraded reporter fusion proteins

ABSTRACT

A fusion polypeptide comprising a protein of interest which has a reduced half-life of expression, and a nucleic acid molecule encoding the fusion polypeptide, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of U.S.application Serial No. 60/411,070, filed Sep. 16, 2002 and U.S.application Ser. No. 60/412,268, filed Sep. 20, 2002, the disclosures ofwhich are incorporated by reference herein.

FIELD OF THE INVENTION

[0002] This invention relates to the field of biochemical assays andreagents. More specifically, this invention relates to modified reporterproteins, e.g., fluorescent reporter proteins, and to methods for theiruse.

BACKGROUND OF THE INVENTION

[0003] Luciferases are enzymes that catalyze the oxidation of asubstrate (luciferin) with the concomitant release of photons of light.Luciferases have been isolated from numerous species, includingColeopteran arthropods and many sea creatures. Because it is easilydetectable and its activity can be quantified with high precision,luciferase/luciferin enzyme/substrate pairs have been used widely tostudy gene expression and protein localization. Unlike another reporterprotein, green fluorescent protein (GFP), which requires up to 30minutes to form a chromophore, the products of luciferases can bedetected immediately upon completion of synthesis of the polypeptidechain (if substrate and oxygen are also present). As luciferase is auseful reporter in numerous species and in a wide variety of cells,luciferases are ideal for monitoring gene up-regulation. However, thestability of native luciferases and native GFP can functionally maskreliable detection of gene down-regulation.

[0004] Protein degradation is necessary to rid cells of damaged andnon-functioning proteins. Intracellular degradation of proteins is ahighly selective process that allows some proteins to survive for hoursor days, while other proteins survive for only minutes, inside the cell.In recent years, the processes controlling protein degradation havebecome an important area of study, further prompted perhaps by reportsthat the failure of key components in protein degradation can becausative in human disease (Bence et al., 2001; McNaught et al., 2001).

[0005] Protein degradation is not limited to the removal of damaged orotherwise abnormal proteins, as a number of regulatory circuits involveproteins with short half-lives (relatively “unstable” proteins). Forexample, proteolysis plays an important regulatory role in many cellularprocesses including metabolic control, cell cycle progression, signaltransduction and transcription (Hicke, 1997; Joazeiro et al., 1999;Murray et al., 1989; Salghetti et al., 2001). A great part of selectiveprotein degradation in eukaryotes appears to be carried out in theproteosome, an ATP-dependent multi-protein complex. In many degradationpathways, the covalent conjugation of ubiquitin, a 76 amino acidpolypeptide, to proteins destined for degradation, precedes degradationin the proteosome (Hershko et al., 1992).

[0006] Omithine decarboxylase (ODC) is an enzyme which is critical inthe biosynthesis of polyamines and is known to have a very shortcellular half-life. In fact, murine omithine decarboxylase (mODC) is oneof most short-lived proteins, with a half-life of about 30 minutes (seeGhoda et al., 1989; Ghoda et al. 1992). Rapid degradation of ODC hasbeen attributed to the unique composition of its C-terminus whichincludes a “PEST” sequence (Rogers et al., 1989; Reichsteiner, 1990). APEST sequence contains a region enriched with proline (P), glutamic acid(E), serine (S), and threonine (T), that is often flanked by basic aminoacids, lysine, arginine, or histidine. The PEST sequence targets thePEST containing protein towards the 26S proteosome without priorubiquitinization (Gilon et al., 1998; Leclerc et al., 2000; Corish etal., 1999; Li et al., 1998; Li et al., 2000). Deletion of the C-terminalPEST containing region from mODC prevents its rapid degradation (Ghodaet al., 1989). In contrast to mODC, the ODC of Trypanosoma brucei(TbODC) does not have a PEST sequence, and is long-lived and quitestable when expressed in mammalian cells (Ghoda et al. 1990). However,fusion of the C-terminus of mODC to TbODC results in an unstableprotein.

[0007] One group has developed destabilized reporter proteins by fusingthe coding sequence of the reporter with a destabilization sequencewhich either destabilizes mRNA encoding the reporter or destabilizes thereporter polypeptide. For instance, the C-terminal region of ODC (C-ODC)has been shown to reduce the half-life of GFP from about 26 hours toabout 9.8 hours (Corish, 1999) and, when a mutant form of C-ODC wasused, to less than 2 hours (Li, 1998; Li, 2000). Moreover, a PESTsequence has been shown to reduce the half-life of firefly luciferasefrom about 3.68 hours to about 0.84 hours (Leclerc et al., 2000). Fan etal. (1997) found that the presence of an AU-rich region from a herpesvirus RNA conferred instability to that RNA as well as to heterologousRNAs, thereby destabilizing the mRNA.

[0008] Peptide signals other than C-ODC that have been used fordestabilization of proteins include the cyclin destruction box (Corishet al., 1999; King et al., 1996), the PEST-rich C-terminal region ofcyclin (Mateus et al., 2000), CL peptides, e.g., CLI (Gilon et al.,1998; Bence et al., 2001) and N-degron. Although all of these signalsdirect proteins containing them towards degradation by the proteosome,the pathways followed by these proteins before they reach the proteosomemay be different. For instance, degradation of ODC occurs in the 26Sproteosome in the absence of prior ubiquitination (Bercovich et al.,1989; Murakami et al., 1992), while CL peptides channel proteins fordegradation via a pathway that depends upon the presence ofubiquitin-conjugating enzymes (Gilon et al., 1998). Degradation ofcyclins is also a ubiquitination-dependent process (Glotzer et al.,1991). Nevertheless, cyclins become unstable only when cells exitmitosis (Hunt et al., 1992).

[0009] More than fifteen years ago Bachmir et al. (1986) described theN-degron degradation signal. These authors reported that certain aminoacid residues, if positioned at the N-terminus of the protein, canstimulate the use of internal protein lysine residues by ubiquitinligase as a point for attachment of ubiquitin. As a result suchN-terminal residues (called destabilizing residues) caused dramaticdestabilization of the corresponding protein. The relationship betweenthe identity of the N-terminal residue and the in vivo half-life of thecorresponding protein has been referred by these authors as the N-endrule (Varshavsky, 1992). This rule has been extensively studied in yeastwhere species of β-galactosidase with N-terminal Arg, Lys, Phe, Leu,Trp, His, Asp, Asn, Tyr, Gln, Ile or Glu had half-lives of 2-30 minutes.At the same time, β-galactosidase species with any of the other eightamino acid residues had half-lives of more than 20 hours (Varshavsky,1992). The most efficient destabilizing residue with a correspondinghalf-life of 2 minutes was found to be arginine. It has been reportedthat the N-end rule also operates in E. coli, where an arginine residuewas found to be the most effective destabilizing residue (Tobias et al.,1991). In E. Coli, similarly to yeast, positioning of this residue atthe N-terminus of β-galactosidase resulted in a protein that had ahalf-life of 2 minutes instead of more than 10 hours (Varshavsky, 1992;Tobias et al., 1991). Several groups have reported data supporting theexistence of the N-end rule in rabbit, mouse and tobacco (Varshavsky,1992; Reiss et al., 1988; Kwon et al., 1998; Townsend et al., 1988).

[0010] However, what is needed is an improved recombinant reporterprotein, e.g., for use in higher eukaryotes.

SUMMARY OF THE INVENTION

[0011] The invention provides improved gene products, e.g., reporterproteins, with reduced or decreased, e.g., substantially reduced ordecreased, half-lives, of expression, which are useful to determine ordetect gene expression, e.g., up- or down-regulation, to monitorpromoter activity, to reduce cytotoxicity, and to localize proteins Inone embodiment, the invention provides an isolated nucleic acid molecule(polynucleotide) comprising a nucleic acid sequence encoding a fusionpolypeptide comprising a reporter protein, e.g., a luciferase, GFP,chloramphenicol acetyltransferase, beta-glucuronidase orbeta-galactosidase, which nucleic acid molecule comprises at least twoheterologous destabilization sequences, e.g., encoding at least twoheterologous protein destabilization sequences, or encoding at least oneheterologous protein destabilization sequence and comprising at leastone heterologous mRNA destabilization sequence. As used herein, a“heterologous” destabilization sequence is one which is not found in thewild-type gene for the reporter protein employed in the fusionpolypeptide. The presence of one or more destabilization sequences in anucleic acid molecule of the invention which is introduced to a hostcell or to an in vitro transcription/translation mixture, results inreporter activity (expression) that is reduced or decreased, e.g., asubstantially reduced or decreased half-life of reporter expression,relative to the reporter activity for a corresponding reporter proteingene that lacks one or more of the destabilization sequences. Forexample, the presence of one or more protein destabilization sequencesin a fusion polypeptide encoded by a nucleic acid molecule of theinvention results in a reduction or decrease in the half-life of thefusion polypeptide relative to a corresponding protein which lacks thedestabilization sequence(s). The presence of one or more RNAdestabilization sequences in a nucleic acid molecule of the inventionresults in a reduction or decrease in the half-life of the mRNAtranscribed from that nucleic acid molecule relative to a nucleic acidmolecule which lacks the destabilization sequence(s). Preferably, thenucleic acid molecule of the invention comprises sequences which havebeen optimized for expression in mammalian cells, and more preferably,in human cells (see, e.g., WO 02/16944 which discloses methods tooptimize sequences for expression in a cell of interest). For instance,nucleic acid molecules may be optimized for expression in eukaryoticcells by introducing a Kozak sequence and/or one or more introns, and/orby altering codon usage to codons employed more frequently in one ormore eukaryotic organisms, e.g., codons employed more frequently in aneukaryotic host cell to be transformed with the nucleic acid molecule.

[0012] A protein destabilization sequence includes one or more aminoacid residues, which, when present at the N-terminus or C-terminus of aprotein of interest, reduces or decreases, e.g., having a reduction ordecrease in the half-life of the protein of interest of at least 80%,preferably at least 90%, more preferably at least 95% or more, e.g.,99%, relative to a corresponding protein which lacks the proteindestabilization sequence. The presence of the protein destabilizationsequence in a fusion polypeptide preferably does not substantially alterother functional properties of the protein of interest. In oneembodiment, a protein destabilization sequence has less than about 200amino acid residues. A protein destabilization sequence includes, but isnot limited to, a PEST sequence, for example, a PEST sequence fromcyclin, e.g., mitotic cyclins, uracil permease or ODC, a sequence fromthe C-terminal region of a short-lived protein such as ODC, earlyresponse proteins such as cytokines, lymphokines, protooncogenes, e.g.,c-myc or c-fos, MyoD, HMG CoA reductase, S-adenosyl methioninedecarboxylase, CL sequences, a cyclin destruction box, N-degron, or aprotein or a fragment thereof which is ubiquitinated in vivo.

[0013] A mRNA destabilization sequence includes two or more nucleotides,which, when present in a mRNA, reduces or decreases, e.g., substantiallyreduces or decreases, for instance, having a reduction or decrease inthe half-life of the mRNA encoding a protein of interest of at least20%, including 50%, 70% or greater, e.g., 90% or 99%, relative to a mRNAthat lacks the mRNA destabilization sequence and encodes thecorresponding protein. In one embodiment, a mRNA destabilizationsequence has less than about 100 nucleotides. A mRNA destabilizationsequence includes, but is not limited to, a sequence present in the 3′UTR of a mRNA which likely forms a stem-loop, one or more AUUUA orUUAUUUAUU sequences, including the 3′ UTR of the bradykinin B1 receptorgene.

[0014] In one embodiment, the nucleic acid molecule is present in avector, e.g., a plasmid. In one embodiment, the nucleic acid moleculeencodes a destabilized fusion polypeptide comprising a reporter protein,which nucleic acid molecule comprises SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:49, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79; SEQ ID NO:80, or afragment thereof that encodes a fusion polypeptide with substantiallythe same activity as the corresponding full-length fusion polypeptide.As used herein, “substantially the same activity” is at least about 70%,e.g., 80%, 90% or more, the activity of a corresponding full-lengthfusion polypeptide.

[0015] As described herein, the combination of two protein degradation(CL1 and mODC) sequences in the same luciferase fusion polypeptideresulted in a reduction of the half-life of firefly luciferase to about30 minutes and Renilla luciferase to about 20 minutes. Also, N-degronand mODC complemented each other in that the combination of these twodegradation signals in the same protein resulted in a substantialincrease in the rate of degradation of the corresponding protein.Moreover, introduction of a mRNA destabilization 3′ to the open readingframe for the luciferase fusion polypeptide decreased the half-life ofluciferase expression by destabilizing sequence transcription. Further,the combination of a mRNA and a protein destabilization sequence wasshown to be effective in at least 3 different cells (HeLa, CHO and 293cells) in shortening the expression of two different luciferaseproteins. In addition, the presence of mammalian cell-optimizedsequences for a fusion polypeptide of the invention, in cellstransfected with a plasmid comprising those sequences, enhanced theamount of light emitted by those cells as a result of the more efficienttranslation of RNA encoding the fusion polypeptide. Thus, the presenceof optimized sequences including codon optimized sequences in a nucleicacid molecule encoding a fusion polypeptide of the invention, e.g.,optimized sequences for the reporter protein, optimized sequences forthe protein destabilization signal(s), or both, can yield an enhancedsignal.

[0016] In one embodiment, the nucleic acid molecule comprises a nucleicacid sequence encoding a fusion polypeptide comprising at least one andpreferably at least two heterologous protein destabilization signals,which fusion polypeptide has a half-life that is substantially reducedor decreased, e.g., having at least a 80%, preferably at least a 90%,more preferably at least a 95% or more, e.g., 99%, reduction or decreasein half-life, relative to the half-life of a corresponding wild-typeprotein, and/or emits more light as a result of the optimization of thenucleic acid sequences for expression in a desired cell relative to afusion polypeptide encoded by sequences which are not optimized forexpression in that cell. In one embodiment, the reporter protein is aluciferase, for instance, a Coleopteran or anthozoan luciferase such asa firefly luciferase or a Renilla luciferase, and the luciferase fusionpolypeptide includes at least one heterologous protein destabilizationsequence and has a substantially reduced half-life relative to acorresponding wild-type (native or recombinant) luciferase. Preferably,optimized nucleic acid sequences encoding at least the reporter proteinare employed, as those optimized sequences can increase the strength ofthe signal for destabilized reporter proteins.

[0017] In another embodiment, the nucleic acid molecule comprises atleast one heterologous mRNA destabilization sequence and encodes afusion polypeptide comprising at least one heterologous proteindestabilization sequence. Preferably, the mRNA destabilization sequenceis 3′ to the nucleic acid sequence encoding the fusion polypeptide. Inone embodiment, the expression of the fusion polypeptide is reducedrelative to a polypeptide encoded by a nucleic acid molecule which lacksthe heterologous destabilization sequences.

[0018] In one embodiment, the nucleic acid molecule comprises a nucleicacid sequence comprising an open reading frame for a reporter proteinand at least one heterologous destabilization sequence, wherein amajority of codons in the open reading frame for the reporter proteinare optimized for expression in a particular host cell, e.g., amammalian cell such as a human cell. The presence of codon optimizedsequences in the nucleic acid molecule can compensate for reducedexpression from a corresponding nucleic acid molecule which is not codonoptimized.

[0019] The invention further includes a vector and host cell comprisinga nucleic acid molecule of the invention and kits comprising the nucleicacid molecule, vector or host cell. In particular the invention providesa stable cell line that expresses a rapid turnover reporter protein withan enhanced signal relative to a corresponding stable cell line thatexpresses a corresponding nondestabilized reporter protein.

[0020] A rapid turnover (destabilized) reporter protein such asluciferase can be used in applications where currently availablereporter proteins with half-lives of expression at least several hourscannot, such as, as a genetic reporter for analyzing transcriptionalregulation and/or cis-acting regulatory elements, as a tool foridentifying and analyzing degradation domains of short-lived proteins orto accelerate screening of efficacious compounds. Cells containing aregulatable vector of the invention respond more quickly to induction orrepression and show enhanced activation relative to cells containing avector expressing a corresponding unmodified, e.g., wild-type, reporterprotein. Moreover, the presence of a vector of the invention in hostcells used for screening is advantageous in that those cells are lesssensitive to impaired cell growth or to modification or loss of thevector, and allows for more precise quantification of signal.

[0021] Hence, the present invention also provides an expression cassettecomprising the nucleic acid sequence of the invention and a vectorcapable of expressing the nucleic acid sequence in a host cell.Preferably, the expression cassette comprises a promoter, e.g., aconstitutive or regulatable promoter, operably linked to the nucleicacid sequence. In one embodiment of the vector, the expression cassettecontains an inducible promoter. Also provided is a host cell, e.g., aneukaryotic cell such as a plant or vertebrate cell, e.g., a mammaliancell, including but not limited to a human, non-human primate, canine,feline, bovine, equine, ovine or rodent (e.g., rabbit, rat, ferret ormouse) cell, and a kit which comprises the nucleic acid molecule,expression cassette or vector of the invention.

[0022] In another aspect of the invention, there is provided a method oflabeling cells with a fusion polypeptide of the invention. In thismethod, a cell is contacted with a vector comprising a promoter, e.g., aregulatable promoter, and a nucleic acid sequence encoding a fusionpolypeptide comprising a protein of interest such as a reporter proteinwith a substantially decreased half-life of expression relative to acorresponding wild-type protein. In one embodiment, a transfected cellis cultured under conditions in which the promoter induces transientexpression of the fusion polypeptide, which provides a transientreporter label.

BRIEF DESCRIPTION OF THE FIGURES

[0023]FIG. 1A. Lysates of CHO cells containing plasmid pwtLuc1 (lane 2),pUbiq(Y)Luc19 (lane 3) or pLuc-PESTIO (lane 4), or a CHO lysate withoutplasmid (lane 5), were separated on 4-20% SDS-PAGE, transferred on to anImmobilonP membrane and luciferase species were detected with rabbitanti-firefly luciferase and anti-rabbit antibodies conjugated withalkaline phosphatase. Lane 1 corresponds to See Blue Pre-StainedStandard from Invitrogen.

[0024]FIG. 1B. Proteins translated with wheat germ extracts containingmRNA obtained using plasmid pwtLuc1 (lane 1) or pETUbiqLuc (lane 2), orwithout external mRNA (lane 3), were separated on 4-20% SDS-PAGE and theproteins visualized by autoradiography.

[0025]FIG. 1C. TNT® T7 Coupled Reticulocyte Lysates containing plasmidpETwtLuc1 (lane 1), pT7Ubiq(Y)Luc19.2 (lane 2), pT7 Ubiq(E)Luc19.1 (lane3) or pT7Luc-PEST10 (lane 4), were separated on 4-20% SDS-PAGE and theproteins visualized by autoradiography.

[0026]FIG. 2. Plasmids encoding wild-type firefly luciferase and fusionproteins comprising firefly luciferase were expressed in TNT® T7 CoupledReticulocyte Lysate System. Specific activity was determined as theratio between total luciferase activity accumulated in each mixture andthe amount of [³H]-Leucine incorporated in each protein.

[0027]FIG. 3. Cells transiently transfected with plasmids encodingwild-type firefly luciferase (pwtLuc1), a ubiquitin-luciferase fusionprotein (pUbiq(Y)Luc19 and pT7Ubiq(Y)Luc19.2), or a fusion proteincomprising firefly luciferase and a mutant form of C-ODC (mODC)(pLuc-PEST10) were treated with cycloheximide (100 μg/ml) for differentperiods of time. Upon completion of incubation, and to define stability,cells were lysed, and accumulated luciferase activity was determinedusing a MLX Microtiter Plate Luminometer.

[0028]FIG. 4. CHO (A), COS-7 (B), and HeLa (C) cells, transfected withubiquitin-luciferase fusion protein encoding plasmids, were treated withcycloheximide for different periods of time. Cellular luminescence wasmeasured to determine the stability of the corresponding proteins.Control cells that had not been treated with cycloheximide were used todetermine background luciferase activity.

[0029]FIG. 5. The partial amino acid sequence of ubiquitin-luciferasefusion proteins was evaluated in establishing the relative importance ofthe N-terminal residue in determining protein half-life. Shadowed/boxedareas mark ubiquitin and luciferase sequences. Thick lines mark theposition of deletions.

[0030]FIG. 6. CHO (A) and COS-7 (B) cells were transiently transfectedwith plasmids encoding either wild-type firefly luciferase (pwtLuc1) orubiquitin-luciferase fusion proteins with different N-terminalluciferase amino acid residues. Twenty-four hours after transfection,the cells were treated with cycloheximide (100 μg/ml) for differentperiods of time and, upon completion of incubation, luminescence ofaccumulated luciferase was measured.

[0031]FIG. 7. HeLa cells were transfected with plasmids encodingwild-type luciferase (pwtLuc1), a fusion protein comprising luciferaseand mODC (pLuc-PEST10), or a fusion protein comprising ubiquitin,firefly luciferase, and mODC (pUbiq(Y)Luc-PEST5, pUbiq(R)Luc-PEST12,pT7Ubiq(E)Luc-PEST23 and pT7Ubiq(E)hLuc+PEST80). Twenty-four hours aftertransfection, the cells were treated with cycloheximide (100 μg/ml) fordifferent periods of time. Cellular luminescence was measured todetermine the stability of the corresponding luciferase (A). Controlcells that had not been treated with cycloheximide were used to comparethe luciferase activity of different constructs (B).

[0032]FIG. 8. CHO cells were transiently transfected with variousplasmids. Twenty-four hours post-transfection, the cells were treatedwith cycloheximide (100 μg/ml) for different periods of time. Afterincubation, luminescence due to accumulated luciferase was measured.Control cells that had not been treated with cycloheximide were used todetermine background luciferase activity.

[0033]FIG. 9. Comparison of luciferase fusion protein properties in atet inducible system after doxycycline (2 μg/ml) (A) or cycloheximide(100 μg/ml) (B) treatment. Luminescence data from control cells that hadnot been treated with either doxycycline or cycloheximide are depictedin panel C.

[0034] FIGS. 10A-B. Comparison of luciferase fusion protein propertiesRenilla luciferase (A) and firefly luciferase (B) in a heat shockinducible system.

[0035]FIG. 11. Schematic of selected vectors.

[0036] FIGS. 12A-B. Induction of luminescence in D293 cells transientlytransfected with Renilla luciferase vectors with multiple CREs,forskolin (10 μM) and isoproterenol (0.25 μM).

[0037] FIGS. 13A-B. Luminescence profiles of hCG-D293 cells transientlytransfected with vectors encoding stable and destabilized versions offirefly luciferase. Cells were treated with isoproterenol (1 μM) andRo-20-1724 (100 μM) or isoproterenol (1 μM) and Ro-20-1724 (100 μM)followed by treatment with human chorionic gonadotropin (hCG) (10 ng/ml)and Ro-20-1724 (100 μM). Arrows indicate time points when chemicals wereadded to the cell cultures.

[0038]FIG. 14. Luminescence versus fold induction in D293 cells stablytransfected with destabilized vectors. Cells were treated with forskolin(10 μM) for 7 hours or incubated in forskolin-free media. All vectorswere under the control of a cAMP regulated promoter.

[0039]FIG. 15. Fold induction by isoproterenol and prostaglandin E1(PGE1) in 293 cells transfected with codon optimized firefly or Renillaluciferase in conjunction with destabilization sequences in a CREsystem. (A)-(B): PGE1 added 24 hours after Iso/Ro; (C)-(D): PGE1 added 6hours after Iso/Ro.

[0040]FIG. 16. Fold induction by isoproterenol in 293 cells transfectedwith either red (CBR) (B) or green (CBG) (A) click beetle sequences inconjunction with destabilization sequences in a CRE system

DETAILED DESCRIPTION OF THE INVENTION

[0041] Definitions

[0042] The term “nucleic acid molecule”, “gene” or “nucleic acidsequence” as used herein, refers to nucleic acid, DNA or RNA, thatcomprises coding sequences necessary for the production of a polypeptideor protein precursor. The polypeptide can be encoded by a full-lengthcoding sequence or by any portion of the coding sequence, as long as thedesired protein activity is retained.

[0043] A “nucleic acid”, as used herein, is a covalently linked sequenceof nucleotides in which the 3′ position of the pentose of one nucleotideis joined by a phosphodiester group to the 5′ position of the pentose ofthe next, and in which the nucleotide residues (bases) are linked inspecific sequence, i.e., a linear order of nucleotides. A“polynucleotide”, as used herein, is a nucleic acid containing asequence that is greater than about 100 nucleotides in length. An“oligonucleotide” or “primer”, as used herein, is a short polynucleotideor a portion of a polynucleotide. An oligonucleotide typically containsa sequence of about two to about one hundred bases. The word “oligo” issometimes used in place of the word “oligonucleotide”.

[0044] Nucleic acid molecules are said to have a “5′-terminus” (5′ end)and a “3′-terminus” (3′ end) because nucleic acid phosphodiesterlinkages occur to the 5′ carbon and 3′ carbon of the pentose ring of thesubstituent mononucleotides. The end of a polynucleotide at which a newlinkage would be to a 5′ carbon is its 5′ terminal nucleotide. The endof a polynucleotide at which a new linkage would be to a 3′ carbon isits 3′ terminal nucleotide. A terminal nucleotide, as used herein, isthe nucleotide at the end position of the 3′- or 5′-terminus.

[0045] DNA molecules are said to have “5′ends” and “3′ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides referred to as the“5′end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring.

[0046] As used herein, a nucleic acid sequence, even if internal to alarger oligonucleotide or polynucleotide, also may be said to have 5′and 3′ ends. In either a linear or circular DNA molecule, discreteelements are referred to as being “upstream” or 5′ of the “downstream”or 3′ elements. This terminology reflects the fact that transcriptionproceeds in a 5′ to 3′ fashion along the DNA strand. Typically, promoterand enhancer elements that direct transcription of a linked gene (e.g.,open reading frame or coding region) are generally located 5′ orupstream of the coding region. However, enhancer elements can exerttheir effect even when located 3′ of the promoter element and the codingregion. Transcription termination and polyadenylation signals arelocated 3′ or downstream of the coding region.

[0047] The term “codon” as used herein, is a basic genetic coding unit,consisting of a sequence of three nucleotides that specify a particularamino acid to be incorporation into a polypeptide chain, or a start orstop signal. The term “coding region” when used in reference tostructural genes refers to the nucleotide sequences that encode theamino acids found in the nascent polypeptide as a result of translationof a mRNA molecule. Typically, the coding region is bounded on the 5′side by the nucleotide triplet “ATG” which encodes the initiatormethionine and on the 3′ side by a stop codon (e.g., TAA, TAG, TGA). Insome cases the coding region is also known to initiate by a nucleotidetriplet “TTG”.

[0048] By “protein” and “polypeptide” is meant any chain of amino acids,regardless of length or post-translational modification (e.g.,glycosylation or phosphorylation). The nucleic acid molecules of theinvention may also encode a variant of a naturally-occurring protein orpolypeptide fragment thereof. Preferably, such a protein polypeptide hasan amino acid sequence that is at least 85%, preferably 90%, and mostpreferably 95% or 99% identical to the amino acid sequence of thenaturally-occurring (native or wild-type) protein from which it isderived.

[0049] Polypeptide molecules are said to have an “amino terminus”(N-terminus) and a “carboxy terminus” (C-terminus) because peptidelinkages occur between the backbone amino group of a first amino acidresidue and the backbone carboxyl group of a second amino acid residue.The terms “N-terminal” and “C-terminal” in reference to polypeptidesequences refer to regions of polypeptides including portions of theN-terminal and C-terminal regions of the polypeptide, respectively. Asequence that includes a portion of the N-terminal region of apolypeptide includes amino acids predominantly from the N-terminal halfof the polypeptide chain, but is not limited to such sequences. Forexample, an N-terminal sequence may include an interior portion of thepolypeptide sequence including bases from both the N-terminal andC-terminal halves of the polypeptide. The same applies to C-terminalregions. N-terminal and C-terminal regions may, but need not, includethe amino acid defining the ultimate N-terminus and C-terminus of thepolypeptide, respectively.

[0050] The term “wild-type” as used herein, refers to a gene or geneproduct that has the characteristics of that gene or gene productisolated from a naturally occurring source. A wild-type gene is thatwhich is most frequently observed in a population and is thusarbitrarily designated the “wild-type” form of the gene. In contrast,the term “mutant” refers to a gene or gene product that displaysmodifications in sequence and/or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

[0051] The term “recombinant protein” or “recombinant polypeptide” asused herein refers to a protein molecule expressed from a recombinantDNA molecule. In contrast, the term “native protein” is used herein toindicate a protein isolated from a naturally occurring (i.e., anonrecombinant) source. Molecular biological techniques may be used toproduce a recombinant form of a protein with identical properties ascompared to the native form of the protein.

[0052] The term “fusion polypeptide” refers to a chimeric proteincontaining a protein of interest (e.g., luciferase) joined to aheterologous sequence (e.g., a non-luciferase amino acid or protein).

[0053] The terms “cell,” “cell line,” “host cell,” as used herein, areused interchangeably, and all such designations include progeny orpotential progeny of these designations. By “transformed cell” is meanta cell into which (or into an ancestor of which) has been introduced anucleic acid molecule of the invention, e.g., via transienttransfection. Optionally, a nucleic acid molecule synthetic gene of theinvention may be introduced into a suitable cell line so as to create astably-transfected cell line capable of producing the protein orpolypeptide encoded by the synthetic gene. Vectors, cells, and methodsfor constructing such cell lines are well known in the art. The words“transformants” or “transformed cells” include the primary transformedcells derived from the originally transformed cell without regard to thenumber of transfers. All progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Nonetheless, mutantprogeny that have the same functionality as screened for in theoriginally transformed cell are included in the definition oftransformants.

[0054] Nucleic acids are known to contain different types of mutations.A “point” mutation refers to an alteration in the sequence of anucleotide at a single base position from the wild type sequence.Mutations may also refer to insertion or deletion of one or more bases,so that the nucleic acid sequence differs from the wild-type sequence.

[0055] The term “homology” refers to a degree of complementarity. Theremay be partial homology or complete homology (i.e., identity). Homologyis often measured using sequence analysis software (e.g., SequenceAnalysis Software Package of the Genetics Computer Group. University ofWisconsin Biotechnology Center. 1710 University Avenue. Madison, Wis.53705). Such software matches similar sequences by assigning degrees ofhomology to various substitutions, deletions, insertions, and othermodifications. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid, asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine.

[0056] The term “isolated” when used in relation to a nucleic acid, asin “isolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant with which it is ordinarily associated in its source. Thus,an isolated nucleic acid is present in a form or setting that isdifferent from that in which it is found in nature. In contrast,non-isolated nucleic acids (e.g., DNA and RNA) are found in the statethey exist in nature. For example, a given DNA sequence (e.g., a gene)is found on the host cell chromosome in proximity to neighboring genes;RNA sequences (e.g., a specific mRNA sequence encoding a specificprotein), are found in the cell as a mixture with numerous other mRNAsthat encode a multitude of proteins. However, isolated nucleic acidincludes, by way of example, such nucleic acid in cells ordinarilyexpressing that nucleic acid where the nucleic acid is in a chromosomallocation different from that of natural cells, or is otherwise flankedby a different nucleic acid sequence than that found in nature. Theisolated nucleic acid or oligonucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acidor oligonucleotide is to be utilized to express a protein, theoligonucleotide contains at a minimum, the sense or coding strand (i.e.,the oligonucleotide may be single-stranded), but may contain both thesense and anti-sense strands (i.e., the oligonucleotide may bedouble-stranded).

[0057] The term “isolated” when used in relation to a polypeptide, as in“isolated protein” or “isolated polypeptide” refers to a polypeptidethat is identified and separated from at least one contaminant withwhich it is ordinarily associated in its source. Thus, an isolatedpolypeptide is present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated polypeptides(e.g., proteins and enzymes) are found in the state they exist innature.

[0058] The term “purified” or “to purify” means the result of anyprocess that removes some of a contaminant from the component ofinterest, such as a protein or nucleic acid. The percent of a purifiedcomponent is thereby increased in the sample.

[0059] The term “operably linked” as used herein refer to the linkage ofnucleic acid sequences in such a manner that a nucleic acid moleculecapable of directing the transcription of a given gene and/or thesynthesis of a desired protein molecule is produced. The term alsorefers to the linkage of sequences encoding amino acids in such a mannerthat a functional (e.g., enzymatically active, capable of binding to abinding partner, capable of inhibiting, etc.) protein or polypeptide isproduced.

[0060] The term “recombinant DNA molecule” means a hybrid DNA sequencecomprising at least two nucleotide sequences not normally found togetherin nature.

[0061] The term “vector” is used in reference to nucleic acid moleculesinto which fragments of DNA may be inserted or cloned and can be used totransfer DNA segment(s) into a cell and capable of replication in acell. Vectors may be derived from plasmids, bacteriophages, viruses,cosmids, and the like.

[0062] The terms “recombinant vector” and “expression vector” as usedherein refer to DNA or RNA sequences containing a desired codingsequence and appropriate DNA or RNA sequences necessary for theexpression of the operably linked coding sequence in a particular hostorganism. Prokaryotic expression vectors include a promoter, a ribosomebinding site, an origin of replication for autonomous replication in ahost cell and possibly other sequences, e.g. an optional operatorsequence, optional restriction enzyme sites. A promoter is defined as aDNA sequence that directs RNA polymerase to bind to DNA and to initiateRNA synthesis. Eukaryotic expression vectors include a promoter,optionally a polyadenlyation signal and optionally an enhancer sequence.

[0063] A polynucleotide having a nucleotide sequence encoding a proteinor polypeptide means a nucleic acid sequence comprising the codingregion of a gene, or in other words the nucleic acid sequence encodes agene product. The coding region may be present in either a cDNA, genomicDNA or RNA form. When present in a DNA form, the oligonucleotide may besingle-stranded (i.e., the sense strand) or double-stranded. Suitablecontrol elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers/promoters, splicejunctions, intervening sequences, polyadenylation signals, etc. Infurther embodiments, the coding region may contain a combination of bothendogenous and exogenous control elements.

[0064] The term “transcription regulatory element” or “transcriptionregulatory sequence” refers to a genetic element or sequence thatcontrols some aspect of the expression of nucleic acid sequence(s). Forexample, a promoter is a regulatory element that facilitates theinitiation of transcription of an operably linked coding region. Otherregulatory elements include, but are not limited to, transcriptionfactor binding sites, splicing signals, polyadenylation signals,termination signals and enhancer elements.

[0065] Transcriptional control signals in eukaryotes comprise “promoter”and “enhancer” elements. Promoters and enhancers consist of short arraysof DNA sequences that interact specifically with cellular proteinsinvolved in transcription. Promoter and enhancer elements have beenisolated from a variety of eukaryotic sources including genes in yeast,insect and mammalian cells. Promoter and enhancer elements have alsobeen isolated from viruses and analogous control elements, such aspromoters, are also found in prokaryotes. The selection of a particularpromoter and enhancer depends on the cell type used to express theprotein of interest. Some eukaryotic promoters and enhancers have abroad host range while others are functional in a limited subset of celltypes. For example, the SV40 early gene enhancer is very active in awide variety of cell types from many mammalian species and has beenwidely used for the expression of proteins in mammalian cells. Two otherexamples of promoter/enhancer elements active in a broad range ofmammalian cell types are those from the human elongation factor 1 gene(Uetsuki et al., 1989; Kim et al., 1990; and Mizushima and Nagata, 1990)and the long terminal repeats of the Rous sarcoma virus (Gorman et al.,1982); and the human cytomegalovirus (Boshart et al., 1985).

[0066] The term “promoter/enhancer” denotes a segment of DNA containingsequences capable of providing both promoter and enhancer functions(i.e., the functions provided by a promoter element and an enhancerelement as described above). For example, the long terminal repeats ofretroviruses contain both promoter and enhancer functions. Theenhancer/promoter may be “endogenous” or “exogenous” or “heterologous.”An “endogenous” enhancer/promoter is one that is naturally linked with agiven gene in the genome. An “exogenous” or “heterologous”enhancer/promoter is one that is placed in juxtaposition to a gene bymeans of genetic manipulation (i.e., molecular biological techniques)such that transcription of the gene is directed by the linkedenhancer/promoter.

[0067] The presence of “splicing signals” on an expression vector oftenresults in higher levels of expression of the recombinant transcript ineukaryotic host cells. Splicing signals mediate the removal of intronsfrom the primary RNA transcript and consist of a splice donor andacceptor site (Sambrook et al., 1989). A commonly used splice donor andacceptor site is the splice junction from the 16S RNA of SV40.

[0068] Efficient expression of recombinant DNA sequences in eukaryoticcells requires expression of signals directing the efficient terminationand polyadenylation of the resulting transcript. Transcriptiontermination signals are generally found downstream of thepolyadenylation signal and are a few hundred nucleotides in length. Theterm “poly(A) site” or “poly(A) sequence” as used herein denotes a DNAsequence which directs both the termination and polyadenylation of thenascent RNA transcript. Efficient polyadenylation of the recombinanttranscript is desirable, as transcripts lacking a poly(A) tail areunstable and are rapidly degraded. The poly(A) signal utilized in anexpression vector may be “heterologous” or “endogenous.” An endogenouspoly(A) signal is one that is found naturally at the 3′ end of thecoding region of a given gene in the genome. A heterologous poly(A)signal is one which has been isolated from one gene and positioned 3′ toanother gene. A commonly used heterologous poly(A) signal is the SV40poly(A) signal. The SV40 poly(A) signal is contained on a 237 bp BamHI/Bcl I restriction fragment and directs both termination andpolyadenylation (Sambrook et al., 1989).

[0069] Eukaryotic expression vectors may also contain “viral replicons”or “viral origins of replication.” Viral replicons are viral DNAsequences which allow for the extrachromosomal replication of a vectorin a host cell expressing the appropriate replication factors. Vectorscontaining either the SV40 or polyoma virus origin of replicationreplicate to high copy number (up to 104 copies/cell) in cells thatexpress the appropriate viral T antigen. In contrast, vectors containingthe replicons from bovine papillomavirus or Epstein-Barr virus replicateextrachromosomally at low copy number (about 100 copies/cell).

[0070] The term “in vitro” refers to an artificial environment and toprocesses or reactions that occur within an artificial environment. Invitro environments include, but are not limited to, test tubes and celllysates. The term “in situ” refers to cell culture. The term “in vivo”refers to the natural environment (e.g., an animal or a cell) and toprocesses or reactions that occur within a natural environment.

[0071] The term “expression system” refers to any assay or system fordetermining (e.g., detecting) the expression of a gene of interest.Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used. A wide range ofsuitable mammalian cells are available from a wide range of sources(e.g., the American Type Culture Collection, Rockland, MD). The methodof transformation or transfection and the choice of expression vehiclewill depend on the host system selected. Transformation and transfectionmethods are described, e.g., in Ausubel et al., 1992. Expression systemsinclude in vitro gene expression assays where a gene of interest (e.g.,a reporter gene) is linked to a regulatory sequence and the expressionof the gene is monitored following treatment with an agent that inhibitsor induces expression of the gene. Detection of gene expression can bethrough any suitable means including, but not limited to, detection ofexpressed mRNA or protein (e.g., a detectable product of a reportergene) or through a detectable change in the phenotype of a cellexpressing the gene of interest. Expression systems may also compriseassays where a cleavage event or other nucleic acid or cellular changeis detected.

[0072] All amino acid residues identified herein are in the naturalL-configuration. In keeping with standard polypeptide nomenclature,abbreviations for amino acid residues are as shown in the followingTable of Correspondence. TABLE OF CORRESPONDENCE 1-Letter 3-Letter AMINOACID Y Tyr L-tyrosine G Gly L-glycine F Phe L-phenylalanine M MetL-methionine A Ala L-alanine S Ser L-serine I Ile L-isoleucine L LeuL-leucine T Thr L-threonine V Val L-valine P Pro L-proline K LysL-lysine H His L-histidine Q Gln L-glutamine E Glu L-glutamic acid W TrpL-tryptophan R Arg L-arginine D Asp L-aspartic acid N Asn L-asparagine CCys L-cysteine

[0073] The invention provides compositions comprising nucleic acidmolecules comprising nucleic acid sequences encoding fusionpolypeptides, as well as methods for using those molecules to yieldfusion polypeptides, comprising a protein of interest with a reduced,e.g., a substantially reduced, half-life of expression relative to acorresponding parental (e.g., wild-type) polypeptide. The invention alsoprovides a fusion polypeptide encoded by such a nucleic acid molecule.The invention may be employed to reduce the half-life of expression ofany protein of interest, e.g., the half-life of a reporter protein. Inparticular, the invention provides an isolated nucleic acid moleculecomprising a nucleic acid sequence encoding a fusion polypeptidecomprising a protein of interest and a combination of heterologousdestabilization sequences, e.g., one or more heterologous proteindestabilization sequences and/or one or more heterologous mRNAdestabilization sequences, which results in a substantial reduction inthe half-life of expression of the encoded fusion polypeptide.Heterologous protein destabilization sequences may be at the N-terminusor the C-terminus, or at the N-terminus and the C-terminus of theprotein of interest. A heterologous protein destabilization sequence mayinclude 2 or more, e.g., 3 to 200, or any integer in between 3 and 200,amino acid residues, although not all of the residues in longersequences, e.g., those greater than 5 residues in length, may be capableof destabilizing a linked amino acid sequence. Multiple copies of anyone protein destabilization sequence may also be employed with a proteinof interest. In one embodiment, different protein destabilizationsequences are employed, e.g., a combination of a CL sequence and a PESTsequence. Heterologous mRNA destabilization sequences are preferably 3′to the coding region for a fusion polypeptide of the invention. Aheterologous mRNA destabilization sequence may include 5 or more, e.g.,6 to 100, or any integer in between 6 and 100, nucleotides, although notall of the residues in longer sequences, e.g., those greater than 10nucleotides, may be capable of destabilizing a linked nucleotidesequence. Multiple copies of any one mRNA destabilization sequence maybe employed. In one embodiment, different mRNA destabilization sequencesare employed.

[0074] Optionally, a second polypeptide may be fused to the N-terminusof a fusion polypeptide comprising a protein of interest and aheterologous protein destabilization sequence, e.g., a destabilizationsequence which is present at the N-terminus of the protein of interest.In one embodiment, the second polypeptide is a polypeptide which iscleaved after the C-terminal residue by an enzyme present in a cell orcell extract, yielding a fusion polypeptide comprising a protein ofinterest with a heterologous protein destabilization sequence, e.g., atits N-terminus. In one embodiment, the second polypeptide is ubiquitin.

[0075] In one embodiment, the N-terminal heterologous proteindestabilization sequence is a cyclin destruction box or N-degron. In oneembodiment, the C-terminal heterologous protein destabilization sequenceis a CL peptide, CL1, CL2, CL6, CL9, CL10, CL11, CL12, CL15, CL216, orCL17, SL17 (see Table 1 of Gilon et al., 1998, which is specificallyincorporated by reference herein), a C-ODC or a mutant C-ODC, e.g., asequence such as HGFXXXMXXQXXGTLPMSCAQESGXXRHPAACASARINV (correspondingto residues 423-461 of mODC), wherein one or more of the residues atpositions marked with “X” are not the naturally occurring residue andwherein the substitution results in a decrease in the stability of aprotein having that substituted sequence relative to a protein havingthe nonsubstituted sequence. For instance, a fusion polypeptidecomprising a mutant C-ODC which has a non-conservative substitution atresidues corresponding to residues 426, 427, 428, 430, 431, 433, 434, or448 of ODC, e.g., from proline, aspartic acid or glutamic acid toalanine, can result in a fusion polypeptide with decreased stability,e.g., relative to a fusion polypeptide with a non-substituted C-ODC.

[0076] The invention may be employed with any nucleic acid sequence,e.g., a native sequence such as a cDNA or one which has been manipulatedin vitro, e.g., but is particularly useful for reporter genes as well asother genes associated with the expression of reporter genes, such asselectable markers. Preferred genes include, but are not limited to,those encoding lactamase (P-gal), neomycin resistance (Neo), CAT, GUS,galactopyranoside, GFP, xylosidase, thymidine kinase, arabinosidase andthe like. As used herein, a “marker gene” or “reporter gene” is a genethat imparts a distinct phenotype to cells expressing the gene and thuspermits cells having the gene to be distinguished from cells that do nothave the gene. Such genes may encode either a selectable or screenablemarker, depending on whether the marker confers a trait which one can‘select’ for by chemical means, i.e., through the use of a selectiveagent (e.g., a herbicide, antibiotic, or the like), or whether it issimply a “reporter” trait that one can identify through observation ortesting, i.e., by ‘screening’. Elements of the present disclosure areexemplified in detail through the use of particular marker genes. Ofcourse, many examples of suitable marker genes or reporter genes areknown to the art and can be employed in the practice of the invention.Therefore, it will be understood that the following discussion isexemplary rather than exhaustive. In light of the techniques disclosedherein and the general recombinant techniques which are known in theart, the present invention renders possible the alteration of any gene.

[0077] Exemplary genes include, but are not limited to, a neo gene, aβ-gal gene, a gus gene, a cat gene, a gpt gene, a hyg gene, a hisD gene,a ble gene, a mprt gene, a bar gene, a nitrilase gene, a mutantacetolactate synthase gene (ALS) or acetoacid synthase gene (AAS), amethotrexate-resistant dhfr gene, a dalapon dehalogenase gene, a mutatedanthranilate synthase gene that confers resistance to 5-methyltryptophan (WO 97/26366), an R-locus gene, a β-lactamase gene, a xy/Egene, an α-amylase gene, a tyrosinase gene, a luciferase (luc) gene,(e.g., a Renilla reniformis luciferase gene, a firefly luciferase gene,or a click beetle luciferase (Pyrophorus plagiophthalamus) gene, anaequorin gene, or a green fluorescent protein gene. Included within theterms selectable or screenable marker genes are also genes which encodea “secretable marker” whose secretion can be detected as a means ofidentifying or selecting for transformed cells. Examples include markerswhich encode a secretable antigen that can be identified by antibodyinteraction, or even secretable enzymes which can be detected by theircatalytic activity. Secretable proteins fall into a number of classes,including small, diffusible proteins detectable, e.g., by ELISA, andproteins that are inserted or trapped in the cell membrane.

[0078] In one embodiment, the nucleic acid sequence encoding the fusionpolypeptide is optimized for expression in a particular cell. Forexample, the nucleic acid sequence is optimized by replacing codons inthe wild-type sequence with codons which are preferentially employed ina particular (selected) cell. Preferred codons have a relatively highcodon usage frequency in a selected cell, and preferably theirintroduction results in the introduction of relatively few transcriptionfactor binding sites, and relatively few other undesirable structuralattributes. Thus, the optimized nucleic acid product has an improvedlevel of expression due to improved codon usage frequency, and a reducedrisk of inappropriate transcriptional behavior due to a reduced numberof undesirable transcription regulatory sequences.

[0079] An isolated nucleic acid molecule of the invention which isoptimized may have a codon composition that differs from that of thecorresponding wild-type nucleic acid sequence at more than 30%, 35%, 40%or more than 45%, e.g., 50%, 55%, 60% or more of the codons. Preferredcodons for use in the invention are those which are employed morefrequently than at least one other codon for the same amino acid in aparticular organism and, more preferably, are also not low-usage codonsin that organism and are not low-usage codons in the organism used toclone or screen for the expression of the nucleic acid molecule.Moreover, preferred codons for certain amino acids (i.e., those aminoacids that have three or more codons,), may include two or more codonsthat are employed more frequently than the other (non-preferred)codon(s). The presence of codons in the nucleic acid molecule that areemployed more frequently in one organism than in another organismresults in a nucleic acid molecule which, when introduced into the cellsof the organism that employs those codons more frequently, is expressedin those cells at a level that is greater than the expression of thewild-type or parent nucleic acid sequence in those cells.

[0080] In one embodiment of the invention, the codons that are differentare those employed more frequently in a mammal, while in anotherembodiment the codons that are different are those employed morefrequently in a plant. A particular type of mammal, e.g., human, mayhave a different set of preferred codons than another type of mammal.Likewise, a particular type of plant may have a different set ofpreferred codons than another type of plant. In one embodiment of theinvention, the majority of the codons which differ are ones that arepreferred codons in a desired host cell. Preferred codons for mammals(e.g., humans) and plants are known to the art (e.g., Wada et al.,1990). For example, preferred human codons include, but are not limitedto, CGC (Arg), CTG (Leu), TCT (Ser), AGC (Ser), ACC (Thr), CCA (Pro),CCT (Pro), GCC (Ala), GGC (Gly), GTG (Val), ATC (Ile), ATT (Ile), AAG(Lys), AAC (Asn), CAG (Gln), CAC (His), GAG (Glu), GAC (Asp), TAC (Tyr),TGC (Cys) and TTC (Phe) (Wada et al., 1990). Thus, in one embodiment,synthetic nucleic acid molecules of the invention have a codoncomposition which differs from a wild type nucleic acid sequence byhaving an increased number of the preferred human codons, e.g. CGC, CTG,TCT, AGC, ACC, CCA, CCT, GCC, GGC, GTG, ATC, ATT, AAG, AAC, CAG, CAC,GAG, GAC, TAC, TGC, TTC, or any combination thereof. For example, thenucleic acid molecule of the invention may have an increased number ofCTG or TTG leucine-encoding codons, GTG or GTC valine-encoding codons,GGC or GGT glycine-encoding codons, ATC or ATT isoleucine-encodingcodons, CCA or CCT proline-encoding codons, CGC or CGT arginine-encodingcodons, AGC or TCT serine-encoding codons, ACC or ACT threonine-encodingcodon, GCC or GCT alanine-encoding codons, or any combination thereof,relative to the wild-type nucleic acid sequence. Similarly, nucleic acidmolecules having an increased number of codons that are employed morefrequently in plants, have a codon composition which differs from awild-type or parent nucleic acid sequence by having an increased numberof the plant codons including, but not limited to, CGC (Arg), CTT (Leu),TCT (Ser), TCC (Ser), ACC (Thr), CCA (Pro), CCT (Pro), GCT (Ser), GGA(Gly), GTG (Val), ATC (Ile), ATT (Ile), AAG (Lys), AAC (Asn), CAA (Gln),CAC (His), GAG (Glu), GAC (Asp), TAC (Tyr), TGC (Cys), TTC (Phe), or anycombination thereof (Murray et al., 1989). Preferred codons may differfor different types of plants (Wada et al., 1990).

[0081] A nucleic acid molecule comprising a nucleic acid sequenceencoding a fusion polypeptide of the invention is optionally operablylinked to transcription regulatory sequences, e.g., enhancers, promotersand transcription termination sequences to form an expression cassette.The nucleic acid molecule is introduced to a vector, e.g., a plasmid orviral vector, which optionally a selectable marker gene, and the vectorintroduced to a cell of interest, for example, a plant (dicot ormonocot), fungus, yeast or mammalian cell. Preferred host cells aremammalian cells such as CHO, COS, 293, Hela, CV-1, and NIH3T3 cells.

[0082] The expression of the encoded fusion polypeptide may becontrolled by any promoter, including but not limited to regulatablepromoters, e.g., an inducible or repressible promoter such as the tetpromoter, the hsp70 promoter and a synthetic promoter regulated by CRE.For example, in the tet-regulated system, the luminescent signal for awild-type luciferase dissipated by 16-17 hours while the signal for afusion polypeptide comprising a heterologous destabilization sequencedissipated to a similar level by 4 hours.

[0083] In one embodiment of the invention, the isolated nucleic acidmolecule comprises a nucleic acid sequence encoding a fusion polypeptidecomprising a reporter protein and at least two destabilizationsequences, wherein the nucleic acid sequence is a synthetic sequencecontaining codons preferentially found in a particular organism, e.g.,in plants or humans, and more preferably in highly expressed proteins,for instance, highly expressed human proteins.

[0084] In one preferred embodiment, the invention provides an isolatednucleic acid molecule comprising a nucleic acid sequence encoding afusion polypeptide comprising a luminescent protein, e.g., a luciferase,and a combination of heterologous protein and/or mRNA destabilizationsequences. Preferably, at least the sequence encoding the luminescentprotein is optimized for expression in human cells.

[0085] The invention will be further described by the followingnon-limiting example.

EXAMPLE 1

[0086] Materials and Methods

[0087] Bacterial Cells and Plasmids

[0088]Escherichia coli JM109 cells were used to propagate plasmids.Bacterial cultures were grown routinely in LB broth at 37° C. with theaddition of 100 μg/ml ampicillin or 30 μg/ml kanamycin when required.Extraction and purification of plasmid DNA were performed using PlasmidMaxi Kit (Qiagen).

[0089] pGEM®-T Easy Vector (Promega) was used to clone PCR products.Plasmids pGL3-Basic Vector and pSP-luc+NF Fusion Vector were used as thesource for cDNA encoding firefly luciferase (Promega).

[0090] DNA Modifying Enzymes

[0091] Restriction enzymes AgeI, ApaI, BamHI, BgIII, BstEII, Bst98I,EcoRI, EcoRV, NcoI, NotI, ScaI, XbaI and XmnI as well as T4 DNApolymerase and S1 nuclease were obtained from Promega. Rapid DNALigation Kit and Expand High Fidelity PCR System were supplied byBoehringer Mannheim.

[0092] Oligonucleotides

[0093] Oligonucleotides used for polymerase chain reactions as well asoligonucleotides used for cloning and sequencing are listed in Table 1.All the oligonucleotides were synthesized at Promega.

[0094] A representative number of these DNA constructs are depictedschematically in FIG. 11.

[0095] pGEM-Luc5 was constructed by cloning into pGEM®-T Easy Vector afragment that encodes firefly luciferase, which was amplified frompGL3-Basic Vector using primers LucN and LucC (Table 1).

[0096] pLuc11 was constructed by joining the large NotI-BglII fragmentof plasmid pUbiqGFP23 with the small BgIII-NotI fragment of plasmidpGEM-Luc5.

[0097] pETwtLuc1 is a derivative of plasmid pET28b(+) that contains thesmall NcoI-EcoRV fragment of plasmid pSP-luc+NF Fusion Vector instead ofthe NcoI-Ecl1361II fragment of plasmid pET28b(+).

[0098] pwtLuc1 was generated by joining the large NotI-ScaI fragment ofplasmid pLuc11 with the small ScaI-NotI fragment of plasmid pETwtLuc1.

[0099] pLuc-PEST10 was generated by joining the large NotI-EcoRIfragment of plasmid pLuc11 with the synthetic DNA fragment that encodesa mutant C-terminal region of the mouse omithine decarboxylase (mODC),which synthetic fragment was formed by oligonucleotides PEST-5′,PEST-3′,5′-PEST and 3′-PEST (Table 1).

[0100] pT7LucPEST10 was generated by joining the large BglII-ScaIfragment of plasmid pLuc-PEST10 with the small ScaI-BglII fragment ofplasmid pETwtLuc1.

[0101] pLucΔRI17 was constructed by treatment of plasmid pLuc11 withEcoR1, T4 DNA polymerase and ligase.

[0102] pSPUbiqLuc1 was generated by combining BstEII-linearized DNA ofplasmid pSP-luc+NF Fusion Vector with the fragment of plasmid pUbiqGFP23which encodes ubiquitin. The ubiquitin fragment was prepared using PCRand primers Ubiquitin 5′ wt/BsytEII and Ubiquitin 3′ w/BstEII (Table 1),and subsequent treatment with BstEII.

[0103] pETUbiqLuc was constructed by joining the large NcoI-Ecl136IIfragment of plasmid pETBirA with the small NcoI-EcoRV fragment ofplasmid pSPUbiqLuc1.

[0104] pUbiqLuc15 was prepared by joining the large fragment of plasmidpUbiqGFP23, which was generated by treatment of pUbiqGFP23 with AgeI, S1nuclease and XbaI, with the small fragment of plasmid pGL3 Basic Vector,which was generated by treatment of pGL3-Basic Vector with NcoI, S1nuclease and XbaI.

[0105] pUbiq(Y)Luc 19 was generated by combining the large XbaI-XmnIfragment of plasmid pUbiqGFP23 with the small XbaI-XmnI fragment ofplasmid pSPUbiqLuc1.

[0106] pT7Ubiq(I)Luc19.1, pT7Ubiq(E)Luc19.1 and pT7Ubiq(M)Luc19.2 weregenerated by combining the large BamHI-ApaI fragment of plasmidpUbiq(Y)Luc19 with BamHI-ApaI treated DNA fragments which had beenamplified by PCR using plasmid pUbiqLuc15 and primers Ubi-Luc 5′w/Linker and Ubi-Luc 3′ with linker or Ubi-Luc3′ w/Linker Glu or Ubi-Luc3′ w/Linker Met, accordingly (Table 1).

[0107] pT7Ubiq(Y)Luc19.2 was generated by joining the large BamHI-XmnIfragment of plasmid pT7Ubiq(I)Luc19.1 with the small BamHI-XmnI fragmentof plasmid pUbiq(Y)Luc19.

[0108] pUbiq(R)Luc13 was generated by combining the large BstEII-XmnIfragment of plasmid pUbiq(Y)Luc19 with the BstEII-XmnI treated DNAfragments, which had been amplified by PCR using plasmid pUbiq(Y)Luc19and primers Ubiquitin 5′wt/BsytEII and Ubiq(R) (Table 1).

[0109] pUbiq(A)Luc2, pUbiq(Asp2)Luc 16, pUbiq(F)Luc 10, pUbiq(His2)Luc3,pUbiq(H)Lucl 1, pUbiq(L)Luc23, pUbiq(K)Luc4, pUbiq(N)Luc25,pUbiq(Q)Luc36 and pUbiq(W)Luc16 were constructed by combining the largeBstEII-XmnI fragment of plasmid pUbiq(R)Luc13 with BstEII-XmnI treatedDNA fragments which had been amplified by PCR using plasmidpUbiq(Y)Luc19 and primers Ubiquitin 5′wt/BsytEII and Ala or Asp, or Phe,or His2, or His, or Leu, or Lys, or Asn, or Gln, or Trp, respectively(Table 1).

[0110] pUbiq(H)ΔLuc18 was constructed by treatment of plasmidpUbiq(H)Luc11 with BstEI1, T4 DNA polymerase and ligase.

[0111] pUbiq(E)ΔLuc6 was generated by joining the large ScaI-XmnIfragment of plasmid pUbiq(H)ΔLuc 18 with a ScaI-XmnI treated PCRamplified fragments. The fragments were amplified from plasmidpT7Ubiq(E)Luc19.1 as separate DNA fragments using primers Ubiquitin5′wt/BsytEII and Ubiq(E)de15′ or Ubiq(E)de13′ and LucC (Table 1) andthen those fragments were combined in a separate PCR using primersUbiquitin 5′wt/BsytEII and LucC.

[0112] pT7Ubiq(E)LucPEST23 was generated by joining the largeBst98I-ScaI fragment of plasmid pT7Ubiq(I)Luc19.1 with the smallBst98I-ScaI-fragment of plasmid pLuc-PEST10.

[0113] pUbiq(R)Luc-PEST12 and pUbiq(Y)Luc-PEST5 were generated byjoining the small Bst98I-ScaI fragment of plasmid pLuc-PEST10 with thelarge Bst98I-ScaI fragment of plasmids pUbiq(R)Luc13 and pUbiq(Y)Luc19,accordingly.

[0114] pGEMhLuc+5 was constructed by cloning into pGEM®-T Easy Vector afragment that encodes firefly luciferase, which fragment was amplifiedusing a template with an optimized firefly luciferase sequence andprimers Luc+N and Luc+C (Table 1).

[0115] phLuc+PEST1 was generated by joining the small EcoRI-HindIIIfragment of plasmid pGEMhLuc+5 with the large EcoRI-HindIII fragment ofplasmid pLuc-PEST10.

[0116] pT7Ubiq(E)hLuc+PEST80 was generated by joining the smallBstEII-VspI fragment of plasmid pT7Ubiq(I)Luc19.1 with the largeBstEII-VspI fragment of plasmid phLuc+PEST1.

[0117] A sequence containing the promoter of the human hsp70 gene(P_(hsp70)) was amplified from human chromosomal DNA using PCR andprimers 5′-ATTAATCTGATCAATAAAGGGTTTAAGG (SEQ ID NO:1) and5′-AAAAAGGTAGTGGACTGTCG (SEQ ID NO:2).

[0118] A UTR destabilization sequence was assembled using primers:5′-CTAGATTTATTTATTTATTTCTTCATATGC (SEQ ID NO:3) and5′-AATTGCATATGAAGAAATAAATAAATAAAT (SEQ ID NO:4).

[0119] A BKB destabilization sequence was assembled using primers:5′-AATTGGGAATTAAAACAGCATTGAACCAAGAAGCTTGGCTTTCTTATCAATTCTTTGTGACATAATAAGTT (SEQ ID NO:5) and5′-AACTTATTATGTCACAAAGAATTGATAAGAAAGCCAAGCTTCTTGG TTCAATGCTGTTTTAATTCCC(SEQ ID NO:6).

[0120] A mutant mODC PEST sequence(HGFPPEMEEQAAGTLPMSCAQESGMDRHPAACASARINV (corresponding to resides423-461 of mODC; SEQ ID NO:7) was assembled using primers:5′-AATTCTCATGGCTTCCCGCCGGAGATGGAGGAGCAGGCTGCTGGCA CGCTGCCCATGTCTT (SEQID NO:8), 5′-GTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGATCAATGTGTAA (SEQ ID NO:9),5′-GGCCTTACACATTGATCCTAGCAGAAGCACAGGCTGCAGGGTGAC GGTCCATCCCGCTCTCCT (SEQID NO:10) and 5′-GGGCACAAGACATGGGCAGCGTGCCAGCAGCCTGCTCCTCCATCTCCGGCGGGAAGCCATGAG (SEQ ID NO:11).

[0121] A CL1 sequence (ACKNWFSSLSHFVIHL; SEQ ID NO:12) was assembledusing oligonucleotides:5′-AATTCAAGTGGATCACGAAGTGGCTCAAGCTGCTGAACCAGTTCTT GCAGGCAGACA (SEQ IDNO:13) and 5′-AATTTGTCTGCCTGCAAGAACTGGTTCAGCAGCTTGAGCCACTTCG TGATCCACTTG(SEQ ID NO:14).

[0122] An optimized PEST sequence (hPEST) has the following sequence:(SEQ ID NO:15) CACGGCTTCCCtCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGAtaGaCACCCtGCtGCtTGCGCCAGCGCCAGGATCAACGTCTAA.

[0123] An optimized CL1 and hPEST with a UTR sequence has the followingsequence: (SEQ ID NO:46)GCtTGCAAGAACTGGTTCAGtAGCtTaAGCCACTTtGTGATCCACCTtAACAGCCACGGCTTCCCtCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGAtaGaCACCCtGCtGCtTGCGCCAGCGCCAGGATCAACGTcTAg.

[0124] pGEM-hRL3 was constructed by cloning into pGEMOT Easy Vector aPCR amplified optimized sequence that encodes Renilla luciferase, whichwas amplified from phRL-TK using primers hRLN and hRLC (Table 1).

[0125] phRL-PEST15 was generated by joining the large HindIII-EcoRIfragment of plasmid pLuc-PEST10 with the small HindIII-EcoRI fragment ofplasmid pGEM-hRL3.

[0126] phRLΔR1-PESTI was constructed by treatment of plasmid phRL-PEST15with EcoR1, T4 DNA polymerase and ligase.

[0127] pT7Ubiq(E)hRL-PEST65 and pUbiq(R)hRL-PEST45 were generated byjoining the large BstEII— VspI fragment of plasmid phRL-PEST15 with thesmall BstEII-VspI fragment of plasmids pT7Ubiq(E)Lucl9.1 andpUbiq(R)Lucl3, accordingly.

[0128] pUbiq(A)hRL1, pUbiq(H)hRL1, pUbiq(F)hRL1 were generated byjoining the large Bst98I-BstEII fragment of plasmid phRLAR1-PEST1 withthe small Bst98I -BstEII fragment of plasmids pUbiq(A)Luc2 orpUbiq(His2)Luc3 or pUbiq(F)Luc 10, accordingly.

[0129] pUbiq(E)hRL1 and pUbiq(R)hRL1 were created by joining the largeHindIII-EcoRV fragment of plasmid phRLΔRI-PESTI with the smallHindIII-EcoRV fragment of plasmids pT7Ubiq(E)hRL-PEST65 orpUbiq(R)hRL-PEST45, accordingly.

[0130] pGEM-tetO1 was constructed by cloning into pGEM®OT Easy Vector aPCR amplified sequence that encodes a hCMV minimal promoter withheptamerized upstream tet-operators (Gossen, 1992), which was amplifiedfrom pUHD 10-3 using primers tetO-3′ and tetO-5′ (Table 1).

[0131] ptetO-hRL9 was generated by treatment of plasmid ptetO-hRL1-PEST1 with endonuclease EcoR1, T4 DNA polymerase and ligase.

[0132] ptetO-hRL-PEST1 was generated by joining the large NheI— VspIfragment of plasmid phRL-PEST15 with the small NheI-VspI fragment ofplasmid pGEM-tetO1.

[0133] ptetO-T7Ubiq(E)hRL-PEST15 was generated by joining the largeNheI-VspI fragment of plasmid pT7Ubiq(E)hRL-PEST65 with the smallNheI-VspI fragment of plasmid pGEM-tetO 1.

[0134] ptetO-Ubiq(E)hRL-PEST6 was constructed by treatment of plasmidptetO-T7Ubiq(E)hRL-PEST 15 with XbaI and Eco47111, T4 DNA polymerase andligase.

[0135] ptetO-Ubiq(E)hRL-PEST-UTR13 was created by joining the largeMuni-XbaI fragment of plasmid ptetO-Ubiq(E)hRL-PEST6 with the adaptorformed by oligonucleotides AUUU and anti-AUUU (Table 1).

[0136] ptetO-hRL-PEST-UTR12 was created by joining the large PstI-KpnIfragment of plasmid ptetO-Ubiq(E)hRL-PEST-UTR13 with the small PstI-KpnIfragment of plasmid ptetO-hRL-CL 1-PEST11.

[0137] ptetO-Ubiq(E)hRL-PEST-BKB24 was created by joining the largeMuni-HpaI fragment of plasmid ptetO-T7Ubiq(E)hRL-PEST15 with the adaptorformed by oligonucleotides 3′-BKB1 and 5′-BKBlrev (Table 1).

[0138] ptetO-T7Ubiq(E)hRL-PEST-UTR-BKB8 was generated by joining thelarge NheI-Muni fragment of plasmid ptetO-Ubiq(E)hRL-PEST-BKB24 with thesmall NheI-Muni fragment of plasmid ptetO-Ubiq(E)hRL-PEST-UTR16.

[0139] ptetO-Ubiq(E)hRL-PEST-UTR 16 was generated by joining the largeMunI-XbaI fragment of plasmid ptetO-Ubiq(E)hRL-PEST6 with the DNAfragment formed by oligonucleotides AUUU (SEQ ID NO:3) and Anti-AUUU(SEQ ID NO:4).

[0140] ptetO-hRL-CL1-PEST11 was generated byjoining the large EcoRIfragment of plasmid ptetO-hRL-PEST1 with the DNA fragment formed byoligonucleotides CL1-N-final (SEQ ID NO:64) and Rev-CL1-N-final (SEQ IDNO:65).

[0141] ptetO-hRL-CL1-PEST-UTR1 was generated by joining the largePstI-KpnI fragment of plasmid ptetO-Ubiq(E)hRL-PEST-UTR16 with the smallPstI-KpnI fragment of plasmid ptetO-hRL-CL1-PESTl 1.

[0142] pGEM-Phsp70-3 was constructed by cloning into pGEMOT Easy Vectora PCR amplified sequence which was amplified from human DNA usingprimers hsp70-5′ and hsp70-3′ (Table 1).

[0143] pPhsp70-hRL-PEST 15 was generated by joining the large NheI— VspIfragment of plasmid ptetO-hRL-PEST1 with the small NheI-VspI fragment ofplasmid pGEM-Phsp70-3.

[0144] pPhsp7o-hRL7 was constructed by treatment of plasmidpPhsp7o-hRL-PEST 15 with EcoRI, T4 DNA polymerase and ligase.

[0145] pPhsp70-Ubiq(E)hRL-PEST 1 was generated by joining the largeNheI-VspI fragment of plasmid ptetO-Ubiq(E)hRL-PEST6 with the smallNheI-VspI fragment of plasmid pGEM-Phsp70-3.

[0146] pPhsp70-Ubiq(E)hRL-PEST-UTR10 was generated by joining the largeNheI-VspI fragment of plasmid ptetO-Ubiq(E)hRL-PEST-UTR16 with the smallNheI-VspI fragment of plasmid pGEM-Phsp70-3.

[0147] pPhsp70-T7Ubiq(E)hRL-PEST-BKB5 was generated by joining the largeNheI-VspI fragment of plasmid ptetO-T7Ubiq(E)hRL-PEST-BKB24 with thesmall NheI-VspI fragment of plasmid pGEM-Phsp70-3.

[0148] pPhsp70-T7Ubiq(E)hRL-PEST-UTR-BKB7 was generated by joining thelarge NheI-VspI fragment of plasmid ptetO-T7Ubiq(E)hRL-PEST-UTR-BKB8with the small NheI-VspI fragment of plasmid pGEM-Phsp70-3.

[0149] pLucCL1-25 was generated by joining the large EcoRI-NotI fragmentof plasmid pLuc 1 with the DNA fragment formed by oligonucleotides CL1(SEQ ID NO:62) and Rev-CL1 (SEQ ID NO:63).

[0150] pLucCL1-PEST9 was generated by joining the large EcoRI fragmentof plasmid pLuc-PEST10 with the DNA fragment formed by oligonucleotidesCL1-N-final (SEQ ID NO:64) and Rev-CL1-N-final (SEQ ID NO:65).

[0151] pCL1-Luc1 was generated by joining the large HindIII-BglIIfragment of plasmid pLucΔR117 with the DNA fragment formed byoligonucleotides CL1-N (SEQ ID NO:64) and Rev-CL1-N (SEQ ID NO:65).

[0152] phLuc+CL1-PEST13 was generated by joining the large EcoRIfragment of plasmid phLuc+PEST1 with the DNA fragment formed byoligonucleotides CL1-N-final (SEQ ID NO:64) and Rev-CL1-N-final (SEQ IDNO:65).

[0153] pPhsp70hLuc+PEST2 was generated by joining the large EcoRI-NheIfragment of plasmid phLuc+PEST1 with the small EcoRI-NheI fragment ofplasmid pPhsp70hRL-PEST15.

[0154] pPhsp70hLuc+14 was constructed by treatment of plasmidpPhsp70hLuc+PEST2 with EcoR1, T4 DNA polymerase and ligase.

[0155] pPhsp70hRL-CL1-PEST-UTR4 was generated by joining the largeVspI-NheI fragment of plasmid ptetO-hRL-CL1-PEST-UTR1 with the smallVspI-NheI fragment of plasmid pGEM-Phsp70-3.

[0156] pPhsp70hLuc+CL 1-PEST 12, pPhsp70hLuc+CL1-PEST-UTR5 weregenerated by joining the small EcoRI-NheI fragment of plasmidphLuc+CL1-PEST13 with large EcoRI-NheI fragments of plasmidspPhsp70hRL-PEST15 and pPhsp70hRL-CL1-PEST-UTR4, respectively.

[0157] pPhsp70 MhLuc+27, pPhsp70MhLuc+PEST25, pPhsp70MhLuc+CL1-PEST32and pPhsp70MhLuc+CL1-PEST-UTR 19 were constructed by cloning DNAfragment formed by oligonucleotides N-M and M-C (Table 1) into plasmidspPhsp70hLuc+14, pPhsp70hLuc+PEST2, pPhsp7ohLuc+CL1-PEST12 andpPhsp70hLuc+CL1-PEST-UTR5, respectively, that were treated with BstEIIand BglII.

[0158] phRL-PEST14 was constructed by joining the large EcoRV-NheIfragment of plasmid phRL-PEST 15 with the small EcoRV-NheI fragment ofplasmid phRL-TK.

[0159] pGL3-hRL-PEST3 was constructed by joining the large Bst98I-XbaIfragment of plasmid pGL3-Ubiq(E)hRL-PEST2 with the small Bst98I-XbaIfragment of plasmid phRL-PEST14.

[0160] pGL3-hRL11 was constructed by joining the large Bst98I-EcoRVfragment of plasmid pGL3-hRL-PEST3 with the small Bst98I-EcoRV fragmentof plasmid phRL3.

[0161] pGL3-hRL-CL1-PEST-UTR23 was constructed by joining the largeBst98I-EcoRV fragment of plasmid pGL3-hRL-PEST3 with the smallBst98I-EcoRV fragment of plasmid ptetO-hRL-CL1-PEST-UTR1.

[0162] pGL3-hRL-PEST-UTR6 was constructed by joining the largeBst98I-EcoRV fragment of plasmid pGL3-hRL-PEST3 with the smallBst98I-EcoRV fragment of plasmid ptetO-Ubiq(E)hRL-PEST-UTR16.

[0163] pGL3-hRL-CL1-PEST7 was constructed by joining the largeBst98I-EcoRV fragment of plasmid pGL3-hRL-PEST3 with the smallBst98I-EcoRV fragment of plasmid ptetO-hRL-CL1-PEST11.

[0164] An optimized Renilla luciferase DNA has the following sequence:(SEQ ID NO:47) atggcttccaaggtgtacgaccccgagcaacgcaaacgcatgatcactgggcctcagtggtgggctcgctgcaagcaaatgaacgtgctggactccttcatcaactactatgattccgagaagcacgccgagaacgccgtgatttttctgcatggtaacgctgcctccagctacctgtggaggcacgtcgtgcctcacatcgagcccgtggctagatgcatcatccctgatctgatcggaatgggtaagtccggcaagagcgggaatggctcatatcgcctcctggatcactacaagtacctcaccgcttggttcgagctgctgaaccttccaaagaaaatcatctttgtgggccacgactggggggcttgctggcctttcactactcctacgagcaccaagacaagatcaaggccatcgtccatgctgagagtgtcgtggacgtgatcgagtcctgggacgagtggcctgacatcgaggaggatatcgccctgatcaagagcgaagagggcgagaaaatggtgcttgagaataacttcttcgtcgagaccatgctcccaagcaagatcatgcggaaactggagcctgaggagttcgctgcctacctggagccattcaaggagaagggcgaggttagacggcctaccctctcctggcctcgcgagatccctctcgttaagggaggcaagcccgacgtcgtccagattgtccgcaactacaacgcctaccttcgggccagcgacgatctgcctaagatgttcatcgagtccgaccctgggttcttttccaacgctattgtcgagggagctaagaagttccctaacaccgagttcgtgaaggtgaagggcctccacttcagccaggaggacgctccagatgaaatgggtaagtacatcaagagcttcgtggag cgcgtgctgaagaacgagcagtaa.

[0165] An optimized firefly luciferase DNA has the following sequence:(SEQ ID NO:48) atggccgatgctaagaacattaagaagggccctgctcccttctaccctctggaggatggcaccgctggcgagcagctgcacaaggccatgaagaggtatgccctggtgcctggcaccattgccttcaccgatgcccacattgaggtggacatcacctatgccgagtacttcgagatgtctgtgcgcctggccgaggccatgaagaggtacggcctgaacaccaaccaccgcatcgtggtgtgctctgagaactctctgcagttcttcatgccagtgctgggcgccctgtcatcggagtggccgtggcccctgctaacgacatttacaacgagcgcgagctgctgaacagcatgggcatttctcagcctaccgtggtgttcgtgtctaagaagggcctgcagaagatcctgaacgtgcagaagaagctgcctatcatccagaagatcatcatcatggactctaagaccgactaccagggcttccagagcatgtacacattcgtgacatctcatctgcctcctggcttcaacgagtacgacttcgtgccagagtctttcgacagggacaaaaccattgccctgatcatgaacagctctgggtctaccggcctgcctaagggcgtggccctgcctcatcgcaccgcctgtgtgcgcttctctcacgcccgcgaccctattttcggcaaccagatcatccccgacaccgctattctgagcgtggtgccattccaccacggcttcggcatgttcaccaccctgggctacctgatttgcggctttcgggtggtgctgatgtaccgcttcgaggaggagctgttcctgcgcagcctgcaagactacaaaattcagtctgccctgctggtgccaaccctgttcagcttcttcgctaagagcaccctgatcgacaagtacgacctgtctaacctgcacgagattgcctctggcggcgccccactgtctaaggaggtgggcgaagccgtggccaagcgctttcatctgccaggcatccgccagggctacggcctgaccgagacaaccagcgccattctgattaccccagagggcgacgacaagcctggcgccgtgggcaaggtggtgccattcttcgaggccaaggtggtggacctggacaccggcaagaccctgggagtgaaccagcgcggcgagctgtgtgtgcgcggccctatgattatgtccggctacgtgaataaccctgaggccacaaacgccctgatcgacaaggacggctggctgcactctggcgacattgcctactgggacgaggacgagcacttcttcatcgtggaccgcctgaagtctctgatcaagtacaagggctaccaggtggccccagccgagctggagtctatcctgctgcagcaccctaacattttcgacgccggagtggccggcctgcccgacgacgatgccggcgagctgcctgccgccgtcgtcgtgctggaacacggcaagaccatgaccgagaaggagatcgtggactatgtggccagccaggtgacaaccgccaagaagctgcgcggcggagtggtgttcgtggacgaggtgcccaagggcctgaccggcaagctggacgcccgcaagatccgcgagatcctgatcaaggctaagaaa ggcggcaagatcgccgtgtaa.

[0166] An optimized mutant firefly luciferase DNA has the followingsequence: (SEQ ID NO:49) atggccgatgctaagaacattaagaagggccctgctcccttctaccctctggaggatggcaccgctggcgagcagctgcacaaggccatgaagaggtatgccctggtgcctggcaccattgccttcaccgatgcccacattgaggtggacatcacctatgccgagtacttcgagatgtctgtgcgcctggccgaggccatgaagaggtacggcctgaacaccaaccaccgcatcgtggtgtgctctgagaactctctgcagttcttcatgccagtgctgggcgccctgttcatcggagtggccgtggcccctgctaacgacatttacaacgagcgcgagctgctgaacagcatgggcatttctcagcctaccgtggtgttcgtgtctaagaagggcctgcagaagatcctgaacgtgcagaagaagctgcctatcatccagaagatcatcatcatggactctaagaccgactaccagggcttccagagcatgtacacattcgtgacatctcatctgcctcctggcttcaacgagtacgacttcgtgccagagtctttcgacagggacaaaaccattgccctgatcatgaacagctctgggtctaccggcctgcctaagggcgtggccctgacccatcgcaacgcctgtgtgcgcttctctcacgcccgcgaccctattttcggcaaccagatcatccccgacaccgctattctgagcgtggtgccattccaccacggcttcggcatgttcaccaccctgggctacctgatttgcggctttcgggtggtgctgatgtaccgcttcgaggaggagctgttcctgcgcagcctgcaagactacaaaattcagtctgccctgctggtgccaaccctgttcagcttcttcgctaagagcaccctgatcgacaagtacgacctgtctaacctgcacgagattgcctctggcggcgccccactgtctaaggaggtgggcgaagccgtggccaagcgctttcatctgccaggcatccgccagggctacggcctgaccgagacaaccagcgccattctgattaccccagagggcgacgacaagcctggcgccgtgggcaaggtggtgccattcttcgaggccaaggtggtggacctggacaccggcaagaccctgggagtgaaccagcgcggcgagctgtgtgtgcgcggccctatgattatgtccggctacgtgaataaccctgaggccacaaacgccctgatcgacaaggacggctggctgcactctggcgacattgcctactgggacgaggacgagcacttcttcatcgtggaccgcctgaagtctctgatcaagtacaagggctaccaggtggccccagccgagctggagtctatcctgctgcagcaccctaacattttcgacgccggagtggccggcctgcccgacgacgatgccggcgagctgcctgccgccgtcgtcgtgctggaacacggcaagaccatgaccgagaaggagatcgtggactatgtggccagccaggtgacaaccgccaagaagctgcgcggcggagtggtgttcgtggacgaggtgcccaagggcctgaccggcaagctggacgcccgcaagatccgcgagatcctgatcaaggctaagaaa ggcggcaagatcgccgtgtaa.

[0167] An optimized GFP sequence has the following sequence: (SEQ IDNO:68) ATGGGCGTGATCAAGCCCGACATGAAGATCAAGCTGCGgATGGAGGGCGCCGTGAACGGCCACAAaTTCGTGATCGAGGGCGACGGgAAaGGCAAGCCCTTtGAGGGtAAGCAGACtATGGACCTGACCGTGATCGAGGGCGCCCCCCTGCCCTTCGCtTAtGACATtCTcACCACCGTGTTCGACTACGGtAACCGtGTcTTCGCCAAGTACCCCAAGGACATCCCtGACTACTTCAAGCAGACCTTCCCCGAGGGCTACtcgTGGGAGCGaAGCATGACaTACGAGGACCAGGGaATCTGtATCGCtACaAACGACATCACCATGATGAAGGGtGTGGACGACTGCTTCGTGTACAAaATCCGCTTCGACGGgGTcAACTTCCCtGCtAAtGGCCCgGTgATGCAGCGCAAGACCCTaAAGTGGGAGCCCAGtACCGAGAAGATGTACGTGCGgGACGGCGTaCTGAAGGGCGAtGTtAAtATGGCaCTGCTCtTGGAGGGaGGCGGCCACTACCGCTGCGACTTCAAGACCACCTACAAaGCCAAGAAGGTGGTGCAGCTtCCCGACTACCACTTCGTGGACCACCGCATCGAGATCGTGAGCCACGACAAGGACTACAACAAaGTcAAGCTGTACGAGCACGCCGAaGCCCACAGCGGaCTaCCCCGCCAGGCCggCTAA.

[0168] Other optimized firefly luciferase sequences include hluc+: (SEQID NO: 66) ATGGCCGATGCTAAGAACATTAAGAAGGGCCCTGCTCCCTTCTACCCTCTGGAGGATGGCACCGCTGGCGAGCAGCTGCACAAGGCCATGAAGAGGTATGCCCTGGTGCCTGGCACCATTGCCTTCACCGATGCCCACATTGAGGTGGACATCACCTATGCCGAGTACTTCGAGATGTCTGTGCGCCTGGCCGAGGCCATGAAGAGGTACGGCCTGAACACCAACCACCGCATCGTGGTGTGCTCTGAGAACTCTCTGCAGTTCTTCATGCCAGTGCTGGGCGCCCTGTTCATCGGAGTGGCCGTGGCCCCTGCTAACGACATTTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATTTCTCAGCCTACCGTGGTGTTCGTGTCTAAGAAGGGCCTGCAGAAGATCCTGAACGTGCAGAAGAAGCTGCCTATCATCCAGAAGATCATCATCATGGACTCTAAGACCGACTACCAGGGCTTCCAGAGCATGTACACATTCGTGACATCTCATCTGCCTCCTGGCTTCAACGAGTACGACTTCGTGCCAGAGTCTTTCGACAGGGACAAAACCATTGCCCTGATCATGAACAGCTCTGGGTCTACCGGCCTGCCTAAGGGCGTGGCCCTGCCCCATCGCACCGCCTGTGTGCGCTTCTCTCACGCCCGCGACCCTATTTTCGGCAACCAGATCATCCCCGACACCGCTATTCTGAGCGTGGTGCCATTCCACCACGGCTTCGGCATGTTCACCACCCTGGGCTACCTGATTTGCGGCTTTCGGGTGGTGCTGATGTACCGCTTCGAGGAGGAGCTGTTCCTGCGCAGCCTGCAAGACTACAAAATTCAGTCTGCCCTGCTGGTGCCAACCCTGTTCAGCTTCTTCGCTAAGAGCACCCTGATCGACAAGTACGACCTGTCTAACCTGCACGAGATTGCCTCTGGCGGCGCCCCACTGTCTAAGGAGGTGGGCGAAGCCGTGGCCAAGCGCTTTCATCTGCCAGGCATCCGCCAGGGCTACGGCCTGACCGAGACAACCAGCGCCATTCTGATTACCCCAGAGGGCGACGACAAGCCTGGCGCCGTGGGCAAGGTGGTGCCATTCTTCGAGGCCAAGGTGGTGGACCTGGACACCGGCAAGACCCTGGGAGTGAACCAGCGCGGCGAGCTGTGTGTGCGCGGCCCTATGATTATGTCCGGCTACGTGAATAACCCTGAGGCCACAAACGCCCTGATCGACAAGGACGGCTGGCTGCACTCTGGCGACATTGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGCCTGAAGTCTCTGATCAAGTACAAGGGCTACCAGGTGGCCCCAGCCGAGCTGGAGTCTATCCTGCTGCAGCACCCTAACATTTTCGACGCCGGAGTGGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCTGCCGCCGTCGTCGTGCTGGAACACGGCAAGACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTGACAACCGCCAAGAAGCTGCGCGGCGGAGTGGTGTTCGTGGACGAGGTGCCCAAGGGCCTGACCGGCAAGCTGGACGCCCGCAAGATCCGCGAGATCCTGATCAAGGCTAAGAAAGGCGGCAAGATCGCCGTGTAA;

[0169] and hLuc+(5F2): (SEQ ID NO:49)ATGGCCGATGCTAAGAACATTAAGAAGGGCCCTGCTCCCTTCTACCCTCTGGAGGATGGCACCGCTGGCGAGCAGCTGCACAAGGCCATGAAGAGGTATGCCCTGGTGCCTGGCACCATTGCCTTCACCGATGCCCACATTGAGGTGGACATCACCTATGCCGAGTACTTCGAGATGTCTGTGCGCCTGGCCGAGGCCATGAAGAGGTACGGCCTGAACACCAACCACCGCATCGTGGTGTGCTCTGAGAACTCTCTGCAGTTCTTCATGCCAGTGCTGGGCGCCCTGTTCATCGGAGTGGCCGTGGCCCCTGCTAACGACATTTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATTTCTCAGCCTACCGTGGTGTTCGTGTCTAAGAAGGGCCTGCAGAAGATCCTGAACGTGCAGAAGAAGCTGCCTATCATCCAGAAGATCATCATCATGGACTCTAAGACCGACTACCAGGGCTTCCAGAGCATGTACACATTCGTGACATCTCATCTGCCTCCTGGCTTCAACGAGTACGACTTCGTGCCAGAGTCTTTCGACAGGGACAAAACCATTGCCCTGATCATGAACAGCTCTGGGTCTACCGGCCTGCCTAAGGGCGTGGCCCTGACCCATCGCAACGCCTGTGTGCGCTTCTCTCACGCCCGCGACCCTATTTTCGGCAACCAGATCATCCCCGACACCGCTATTCTGAGCGTGGTGCCATTCCACCACGGCTTCGGCATGTTCACCACCCTGGGCTACCTGATTTGCGGCTTTCGGGTGGTGCTGATGTACCGCTTCGAGGAGGAGCTGTTCCTGCGCAGCCTGCAAGACTACAAAATTCAGTCTGCCCTGCTGGTGCCAACCCTGTTCAGCTTCTTCGCTAAGAGCACCCTGATCGACAAGTACGACCTGTCTAACCTGCACGAGATTGCCTCTGGCGGCGCCCCACTGTCTAAGGAGGTGGGCGAAGCCGTGGCCAAGCGCTTTCATCTGCCAGGCATCCGCCAGGGCTACGGCCTGACCGAGACAACCAGCGCCATTCTGATTACCCCAGAGGGCGACGACAAGCCTGGCGCCGTGGGCAAGGTGGTGCCATTCTTCGAGGCCAAGGTGGTGGACCTGGACACCGGCAAGACCCTGGGAGTGAACCAGCGCGGCGAGCTGTGTGTGCGCGGCCCTATGATTATGTCCGGCTACGTGAATAACCCTGAGGCCACAAACGCCCTGATCGACAAGGACGGCTGGCTGCACTCTGGCGACATTGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGCCTGAAGTCTCTGATCAAGTACAAGGGCTACCAGGTGGCCCCAGCCGAGCTGGAGTCTATCCTGCTGCAGCACCCTAACATTTTCGACGCCGGAGTGGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCTGCCGCCGTCGTCGTGCTGGAACACGGCAAGACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTGACAACCGCCAAGAAGCTGCGCGGCGGAGTGGTGTTCGTGGACGAGGTGCCCAAGGGCCTGACCGGCAAGCTGGACGCCCGCAAGATCCGCGAGATCCTGATCAAGGCTAAGAAAGGCGGCAAGATCGCCGTGTAA.

[0170] An optimized firefly luciferase (hluc+(5f2))-optimized PESTsequence (hluc+(5f2)-hPEST) has the following sequence: (SEQ ID NO:69)atggccgatgctaagaacattaagaagggccctgctcccttctaccctctggaggatggcaccgctggcgagcagctgcacaaggccatgaagaggtatgccctggtgcctggcaccattgccttcaccgatgcccacattgaggtggacatcacctatgccgagtacttcgagatgtctgtgcgcctggccgaggccatgaagaggtacggcctgaacaccaaccaccgcatcgtggtgtgctctgagaactctctgcagttcttcatgccagtgctgggcgccctgttcatcggagtggccgtggcccctgctaacgacatttacaacgagcgcgagctgctgaacagcatgggcatttctcagcctaccgtggtgttcgtgtctaagaagggcctgcagaagatcctgaacgtgcagaagaagctgcctatcatccagaagatcatcatcatggactctaagaccgactaccagggcttccagagcatgtacacattcgtgacatctcatctgcctcctggcttcaacgagtacgacttcgtgccagagtctttcgacagggacaaaaccattgccctgatcatgaacagctctgggtctaccggcctgcctaagggcgtggccctgacccatcgcaacgcctgtgtgcgcttctctcacgcccgcgaccctattttcggcaaccagatcatccccgacaccgctattctgagcgtggtgccattccaccacggcttcggcatgttcaccaccctgggctacctgatttgcggctttcgggtggtgctgatgtaccgcttcgaggaggagctgttcctgcgcagcctgcaagactacaaaattcagtctgccctgctggtgccaaccctgttcagcttcttcgctaagagcaccctgatcgacaagtacgacctgtctaacctgcacgagattgcctctggcggcgccccactgtctaaggaggtgggcgaagccgtggccaagcgctttcatctgccaggcatccgccagggctacggcctgaccgagacaaccagcgccattctgattaccccagagggcgacgacaagcctggcgccgtgggcaaggtggtgccattcttcgaggccaaggtggtggacctggacaccggcaagaccctgggagtgaaccagcgcggcgagctgtgtgtgcgcggccctatgattatgtccggctacgtgaataaccctgaggccacaaacgccctgatcgacaaggacggctggctgcactctggcgacattgcctactgggacgaggacgagcacttcttcatcgtggaccgcctgaagtctctgatcaagtacaagggctaccaggtggccccagccgagctggagtctatcctgctgcagcaccctaacattttcgacgccggagtggccggcctgcccgacgacgatgccggcgagctgcctgccgccgtcgtcgtgctggaacacggcaagaccatgaccgagaaggagatcgtggactatgtggccagccaggtgacaaccgccaagaagctgcgcggcggagtggtgttcgtggacgaggtgcccaagggcctgaccggcaagctggacgcccgcaagatccgcgagatcctgatcaaggctaagaaaggcggcaagatcgccgtgaattctCACGGCTTCCCtCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGAtaGaCACCCtGCtGCtTGCGCCAGCGCCAGGATCAACGTcTAA.

[0171] An optimized firefly luciferase(hluc+(5 f2))-optimizedCL1-optimzed PEST sequence (hluc+(5f2)-hCL1-hPEST) has the followingsequence: (SEQ ID NO: 70) atggccgatgctaagaacattaagaagggccctgctcccttctaccctctggaggatggcaccgctggcgagcagctgcacaaggccatgaagaggtatgccctggtgcctggcaccattgccttcaccgatgcccacattgaggtggacatcacctatgccgagtacttcgagatgtctgtgcgcctggccgaggccatgaagaggtacggcctgaacaccaaccaccgcatcgtggtgtgctctgagaactctctgcagttcttcatgccagtgctgggcgccctgttcatcggagtggccgtggcccctgctaacgacatttacaacgagcgcgagctgctgaacagcatgggcatttctcagcctaccgtggtgttcgtgtctaagaagggcctgcagaagatcctgaacgtgcagaagaagctgcctatcatccagaagatcatcatcatggactctaagaccgactaccagggcttccagagcatgtacacattcgtgacatctcatctgcctcctggcttcaacgagtacgacttcgtgccagagtctttcgacagggacaaaaccattgccctgatcatgaacagctctgggtctaccggcctgcctaagggcgtggccctgacccatcgcaacgcctgtgtgcgcttctctcacgcccgcgaccctattttcggcaaccagatcatccccgacaccgctattctgagcgtggtgccattccaccacggcttcggcatgttcaccaccctgggctacctgatttgcggctttcgggtggtgctgatgtaccgcttcgaggaggagctgttcctgcgcagcctgcaagactacaaaattcagtctgccctgctggtgccaaccctgttcagcttcttcgctaagagcaccctgatcgacaagtacgacctgtctaacctgcacgagattgcctctggcggcgccccactgtctaaggaggtgggcgaagccgtggccaagcgctttcatctgccaggcatccgccagggctacggcctgaccgagacaaccagcgccattctgattaccccagagggcgacgacaagcctggcgccgtgggcaaggtggtgccattcttcgaggccaaggtggtggacctggacaccggcaagaccctgggagtgaaccagcgcggcgagctgtgtgtgcgcggccctatgattatgtccggctacgtgaataaccctgaggccacaaacgccctgatcgacaaggacggctggctgcactctggcgacattgcctactgggacgaggacgagcacttcttcatcgtggaccgcctgaagtctctgatcaagtacaagggctaccaggtggccccagccgagctggagtctatcctgctgcagcaccctaacattttcgacgccggagtggccggcctgcccgacgacgatgccggcgagctgcctgccgccgtcgtcgtgctggaacacggcaagaccatgaccgagaaggagatcgtggactatgtggccagccaggtgacaaccgccaagaagctgcgcggcggagtggtgttcgtggacgaggtgcccaagggcctgaccggcaagctggacgcccgcaagatccgcgagatcctgatcaaggctaagaaaggcggcaagatcgccgtgaattctGCtTGCAAGAACTGGTTCAGtAGCtTaAGCCACTTtGTGATCCACCTtAACAGCCACGGCTTCCCtCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGAtaGaCACCCtGCtGCtTGCGCCAGCGCCAGGATCAACGTcTAg.

[0172] An optimized firefly luciferase (hluc+)-optimized PEST sequence(hluc+-hPEST) has the following sequence: (SEQ ID NO:71)atggccgatgctaagaacattaagaagggccctgctcccttctaccctctggaggatggcaccgctggcgagcagctgcacaaggccatgaagaggtatgccctggtgcctggcaccattgccttcaccgatgcccacattgaggtggacatcacctatgccgagtacttcgagatgtctgtgcgcctggccgaggccatgaagaggtacggcctgaacaccaaccaccgcatcgtggtgtgctctgagaactctctgcagttcttcatgccagtgctgggcgccctgttcatcggagtggccgtggcccctgctaacgacatttacaacgagcgcgagctgctgaacagcatgggcatttctcagcctaccgtggtgttcgtgtctaagaagggcctgcagaagatcctgaacgtgcagaagaagctgcctatcatccagaagatcatcatcatggactctaagaccgactaccagggcttccagagcatgtacacattcgtgacatctcatctgcctcctggcttcaacgagtacgacttcgtgccagagtctttcgacagggacaaaaccattgccctgatcatgaacagctctgggtctaccggcctgcctaagggcgtggccctgcctcatcgcaccgcctgtgtgcgcttctctcacgcccgcgaccctattttcggcaaccagatcatccccgacaccgctattctgagcgtggtgccattccaccacggcttcggcatgttcaccaccctgggctacctgatttgcggctttcgggtggtgctgatgtaccgcttcgaggaggagctgttcctgcgcagcctgcaagactacaaaattcagtctgccctgctggtgccaaccctgttcagcttcttcgctaagagcaccctgatcgacaagtacgacctgtctaacctgcacgagattgcctctggcggcgccccactgtctaaggaggtgggcgaagccgtggccaagcgctttcatctgccaggcatccgccagggctacggcctgaccgagacaaccagcgccattctgattaccccagagggcgacgacaagcctggcgccgtgggcaaggtggtgccattcttcgaggccaaggtggtggacctggacaccggcaagaccctgggagtgaaccagcgcggcgagctgtgtgtgcgcggccctatgattatgtccggctacgtgaataaccctgaggccacaaacgccctgatcgacaaggacggctggctgcactctggcgacattgcctactgggacgaggacgagcacttcttcatcgtggaccgcctgaagtctctgatcaagtacaagggctaccaggtggccccagccgagctggagtctatcctgctgcagcaccctaacattttcgacgccggagtggccggcctgcccgacgacgatgccggcgagctgcctgccgccgtcgtcgtgctggaacacggcaagaccatgaccgagaaggagatcgtggactatgtggccagccaggtgacaaccgccaagaagctgcgcggcggagtggtgttcgtggacgaggtgcccaagggcctgaccggcaagctggacgcccgcaagatccgcgagatcctgatcaaggctaagaaaggcggcaagatcgccgtgaattctcACGGCTTCCCtCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGAtaGaCACCCtGCtGCtTGCGCC AGCGCCAGGATCAACGTcTAA.

[0173] An optimized firefly luciferase (hluc+)-optimized CL1-optimizedPEST sequence (hluc+-hCL1-hPEST) has the following sequence: (SEQ IDNO:72) atggccgatgctaagaacattaagaagggccctgctcccttctaccctctggaggatggcaccgctggcgagcagctgcacaaggccatgaagaggtatgccctggtgcctggcaccattgccttcaccgatgcccacattgaggtggacatcacctatgccgagtacttcgagatgtctgtgcgcctggccgaggccatgaagaggtacggcctgaacaccaaccaccgcatcgtggtgtgctctgagaactctctgcagttcttcatgccagtgctgggcgccctgttcatcggagtggccgtggcccctgctaacgacatttacaacgagcgcgagctgctgaacagcatgggcatttctcagcctaccgtggtgttcgtgtctaagaagggcctgcagaagatcctgaacgtgcagaagaagctgcctatcatccagaagatcatcatcatggactctaagaccgactaccagggcttccagagcatgtacacattcgtgacatctcatctgcctcctggcttcaacgagtacgacttcgtgccagagtctttcgacagggacaaaaccattgccctgatcatgaacagctctgggtctaccggcctgcctaagggcgtggccctgcctcatcgcaccgcctgtgtgcgcttctctcacgcccgcgaccctattttcggcaaccagatcatccccgacaccgctattctgagcgtggtgccattccaccacggcttcggcatgttcaccaccctgggctacctgatttgcggctttcgggtggtgctgatgtaccgcttcgaggaggagctgttcctgcgcagcctgcaagactacaaaattcagtctgccctgctggtgccaaccctgttcagcttcttcgctaagagcaccctgatcgacaagtacgacctgtctaacctgcacgagattgcctctggcggcgccccactgtctaaggaggtgggcgaagccgtggccaagcgctttcatctgccaggcatccgccagggctacggcctgaccgagacaaccagcgccattctgattaccccagagggcgacgacaagcctggcgccgtgggcaaggtggtgccattcttcgaggccaaggtggtggacctggacaccggcaagaccctgggagtgaaccagcgcggcgagctgtgtgtgcgcggccctatgattatgtccggctacgtgaataaccctgaggccacaaacgccctgatcgacaaggacggctggctgcactctggcgacattgcctactgggacgaggacgagcacttcttcatcgtggaccgcctgaagtctctgatcaagtacaagggctaccaggtggccccagccgagctggagtctatcctgctgcagcaccctaacattttcgacgccggagtggccggcctgcccgacgacgatgccggcgagctgcctgccgccgtcgtcgtgctggaacacggcaagaccatgaccgagaaggagatcgtggactatgtggccagccaggtgacaaccgccaagaagctgcgcggcggagtggtgttcgtggacgaggtgcccaagggcctgaccggcaagctggacgcccgcaagatccgcgagatcctgatcaaggctaagaaaggcggcaagatcgccgtgaattctGCtTGCAAGAACTGGTTCAGtAGCtTaAGCCACTTtGTGATCCACCTtAACAGCCACGGCTTCCCtCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGAtaGaCACCCtGCtGCtTGCGCCAGCGCCAGGATCAACGTcTAg.

[0174] An optimized Renilla luciferase-optimized PEST sequence(hRenilla-hPEST) has the following sequence: (SEQ ID NO:73)atggcttccaaggtgtacgaccccgagcaacgcaaacgcatgatcactgggcctcagtggtgggctcgctgcaagcaaatgaacgtgctggactccttcatcaactactatgattccgagaagcacgccgagaacgccgtgatttttctgcatggtaacgctgcctccagctacctgtggaggcacgtcgtgcctcacatcgagcccgtggctagatgcatcatccctgatctgatcggaatgggtaagtccggcaagagcgggaatggctcatatcgcctcctggatcactacaagtacctcaccgcttggttcgagctgctgaaccttccaaagaaaatcatctttgtgggccacgactggggggcttgtctggcctttcactactcctacgagcaccaagacaagatcaaggccatcgtccatgctgagagtgtcgtggacgtgatcgagtcctgggacgagtggcctgacatcgaggaggatatcgccctgatcaagagcgaagagggcgagaaaatggtgcttgagaataacttcttcgtcgagaccatgctcccaagcaagatcatgcggaaactggagcctgaggagttcgctgcctacctggagccattcaaggagaagggcgaggttagacggcctaccctctcctggcctcgcgagatccctctcgttaagggaggcaagcccgacgtcgtccagattgtccgcaactacaacgcctaccttcgggccagcgacgatctgcctaagatgttcatcgagtccgaccctgggttcttttccaacgctattgtcgagggagctaagaagttccctaacaccgagttcgtgaaggtgaagggcctccacttcagccaggaggacgctccagatgaaatgggtaagtacatcaagagcttcgtggagcgcgtgctgaagaacgagcagaattctCACGGCTTCCCtCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGAtaGaCACCCtGCtGCtTGCGCCAGCGC CAGGATCAACGTcTAA.

[0175] An optimized Renilla luciferase-optimized CLlI-optimized PESTsequence (hRenilla-hCLl-hPEST) has the following sequence: (SEQ IDNO:74) atggcttccaaggtgtacgaccccgagcaacgcaaacgcatgatcactgggcctcagtggtgggctcgctgcaagcaaatgaacgtgctggactccttcatcaactactatgattccgagaagcacgccgagaacgccgtgatttttctgcatggtaacgctgcctccagctacctgtggaggcacgtcgtgcctcacatcgagcccgtggctagatgcatcatccctgatctgatcggaatgggtaagtccggcaagagcgggaatggctcatatcgcctcctggatcactacaagtacctcaccgcttggttcgagctgctgaaccttccaaagaaaatcatctttgtgggccacgactggggggcttgtctggcctttcactactcctacgagcaccaagacaagatcaaggccatcgtccatgctgagagtgtcgtggacgtgatcgagtcctgggacgagtggcctgacatcgaggaggatatcgccctgatcaagagcgaagagggcgagaaaatggtgcttgagaataacttcttcgtcgagaccatgctcccaagcaagatcatgcggaaactggagcctgaggagttcgctgcctacctggagccattcaaggagaagggcgaggttagacggcctaccctctcctggcctcgcgagatccctctcgttaagggaggcaagcccgacgtcgtccagattgtccgcaactacaacgcctaccttcgggccagcgacgatctgcctaagatgttcatcgagtccgaccctgggttcttttccaacgctattgtcgagggagctaagaagttccctaacaccgagttcgtgaaggtgaagggcctccacttcagccaggaggacgctccagatgaaatgggtaagtacatcaagagcttcgtggagcgcgtgctgaagaacgagcagaattctGCtTGCAAGAACTGGTTCAGtAGCtTaAGCCACTTtGTGATCCACCTtAACAGCCACGGCTTCCCtCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGAtaGaCACCCtGCtGCtTGCGCCA GCGCCAGGATCAACGTcTAg.

[0176] An optimized Renilla luciferase-optimized CLi-optimized PEST-UTRsequence (hRluc-hCL1-hPEST-UTR) has the following sequence: (SEQ IDNO:75) ATGGCTTCCAAGGTGTACGACCCCGAGCAACGCAAACGCATGATCACTGGGCCTCAGTGGTGGGCTCGCTGCAAGCAAATGAACGTGCTGGACTCCTTCATCAACTACTATGATTCCGAGAAGCACGCCGAGAACGCCGTGATTTTTCTGCATGGTAACGCTGCCTCCAGCTACCTGTGGAGGCACGTCGTGCCTCACATCGAGCCCGTGGCTAGATGCATCATCCCTGATCTGATCGGAATGGGTAAGTCCGGCAAGAGCGGGAATGGCTCATATCGCCTCCTGGATCACTACAAGTACCTCACCGCTTGGTTCGAGCTGCTGAACCTTCCAAAGAAAATCATCTTTGTGGGCCACGACTGGGGGGCTTGTCTGGCCTTTCACTACTCCTACGAGCACCAAGACAAGATCAAGGCCATCGTCCATGCTGAGAGTGTCGTGGACGTGATCGAGTCCTGGGACGAGTGGCCTGACATCGAGGAGGATATCGCCCTGATCAAGAGCGAAGAGGGCGAGAAAATGGTGCTTGAGAATAACTTCTTCGTCGAGACCATGCTCCCAAGCAAGATCATGCGGAAACTGGAGCCTGAGGAGTTCGCTGCCTACCTGGAGCCATTCAAGGAGAAGGGCGAGGTTAGACGGCCTACCCTCTCCTGGCCTCGCGAGATCCCTCTCGTTAAGGGAGGCAAGCCCGACGTCGTCCAGATTGTCCGCAACTACAACGCCTACCTTCGGGCCAGCGACGATCTGCCTAAGATGTTCATCGAGTCCGACCCTGGGTTCTTTTCCAACGCTATTGTCGAGGGAGCTAAGAAGTTCCCTAACACCGAGTTCGTGAAGGTGAAGGGCCTCCACTTCAGCCAGGAGGACGCTCCAGATGAAATGGGTAAGTACATCAAGAGCTTCGTGGAGCGCGTGCTGAAGAACGAGCAGAATTCTGCTTGCAAGAACTGGTTCAGTAGCTTAAGCCACTTTGTGATCCACCTTAACAGCCACGGCTTCCCTCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGACACCCTGCTGCTTGCGCCAGCGCCAGGATCAACGTCTAGGGCGCGGACTTTATTT ATTTATTTCTT

[0177] An optimized firefly luciferase-optimized CL1-optimized PEST-UTRsequence (hluc+-hCL1-hPEST-UTR) has the following sequence: (SEQ IDNO:76) ATGGCCGATGCTAAGAACATTAAGAAGGGCCCTGCTCCCTTCTACCCTCTGGAGGATGGCACCGCTGGCGAGCAGCTGCACAAGGCCATGAAGAGGTATGCCCTGGTGCCTGGCACCATTGCCTTCACCGATGCCCACATTGAGGTGGACATCACCTATGCCGAGTACTTCGAGATGTCTGTGCGCCTGGCCGAGGCCATGAAGAGGTACGGCCTGAACACCAACCACCGCATCGTGGTGTGCTCTGAGAACTCTCTGCAGTTCTTCATGCCAGTGCTGGGCGCCCTGTTCATCGGAGTGGCCGTGGCCCCTGCTAACGACATTTACAACGAGCGCGAGCTGCTGAACAGCATGGGCATTTCTCAGCCTACCGTGGTGTTCGTGTCTAAGAAGGGCCTGCAGAAGATCCTGAACGTGCAGAAGAAGCTGCCTATCATCCAGAAGATCATCATCATGGACTCTAAGACCGACTACCAGGGCTTCCAGAGCATGTACACATTCGTGACATCTCATCTGCCTCCTGGCTTCAACGAGTACGACTTCGTGCCAGAGTCTTTCGACAGGGACAAAACCATTGCCCTGATCATGAACAGCTCTGGGTCTACCGGCCTGCCTAAGGGCGTGGCCCTGCCTCATCGCACCGCCTGTGTGCGCTTCTCTCACGCCCGCGACCCTATTTTCGGCAACCAGATCATCCCCGACACCGCTATTCTGAGCGTGGTGCCATTCCACCACGGCTTCGGCATGTTCACCACCCTGGGCTACCTGATTTGCGGCTTTCGGGTGGTGCTGATGTACCGCTTCGAGGAGGAGCTGTTCCTGCGCAGCCTGCAAGACTACAAAATTCAGTCTGCCCTGCTGGTGCCAACCCTGTTCAGCTTCTTCGCTAAGAGCACCCTGATCGACAAGTACGACCTGTCTAACCTGCACGAGATTGCCTCTGGCGGCGCCCCACTGTCTAAGGAGGTGGGCGAAGCCGTGGCCAAGCGCTTTCATCTGCCAGGCATCCGCCAGGGCTACGGCCTGACCGAGACAACCAGCGCCATTCTGATTACCCCAGAGGGCGACGACAAGCCTGGCGCCGTGGGCAAGGTGGTGCCATTCTTCGAGGCCAAGGTGGTGGACCTGGACACCGGCAAGACCCTGGGAGTGAACCAGCGCGGCGAGCTGTGTGTGCGCGGCCCTATGATTATGTCCGGCTACGTGAATAACCCTGAGGCCACAAACGCCCTGATCGACAAGGACGGCTGGCTGCACTCTGGCGACATTGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGCCTGAAGTCTCTGATCAAGTACAAGGGCTACCAGGTGGCCCCAGCCGAGCTGGAGTCTATCCTGCTGCAGCACCCTAACATTTTCGACGCCGGAGTGGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCTGCCGCCGTCGTCGTGCTGGAACACGGCAAGACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTGACAACCGCCAAGAAGCTGCGCGGCGGAGTGGTGTTCGTGGACGAGGTGCCCAAGGGCCTGACCGGCAAGCTGGACGCCCGCAAGATCCGCGAGATCCTGATCAAGGCTAAGAAAGGCGGCAAGATCGCCGTGAATTCTGCTTGCAAGAACTGGTTCAGTAGCTTAAGCCACTTTGTGATCCACCTTAACAGCCACGGCTTCCCTCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGACACCCTGCTGCTTGCGCCAGCGCCAGGATCAACGTCTAGGGCGCGGACTTTATTTATTT ATTTCTT

[0178] Optimized click beetle sequences include: CBRluc-hPEST SEQ IDNO:77) ATGGTAAAGCGTGAGAAAAATGTCATCTATGGCCCTGAGCCTCTCCATCCTTTGGAGGATTTGACTGCCGGCGAAATGCTGTTTCGTGCTCTCCGCAAGCACTCTCATTTGCCTCAAGCCTTGGTCGATGTGGTCGGCGATGAATCTTTGAGCTACAAGGAGTTTTTTGAGGCAACCGTCTTGCTGGCTCAGTCCCTCCACAATTGTGGCTACAAGATGAACGACGTCGTTAGTATCTGTGCTGAAAACAATACCCGTTTCTTCATTCCAGTCATCGCCGCATGGTATATCGGTATGATCGTGGCTCCAGTCAACGAGAGCTACATTCCCGACGAACTGTGTAAAGTCATGGGTATCTCTAAGCCACAGATTGTCTTCACCACTAAGAATATTCTGAACAAAGTCCTGGAAGTCCAAAGCCGCACCAACTTTATTAAGCGTATCATCATCTTGGACACTGTGGAGAATATTCACGGTTGCGAATCTTTGCCTAATTTCATCTCTCGCTATTCAGACGGCAACATCGCAAACTTTAAACCACTCCACTTCGACCCTGTGGAACAAGTTGCAGCCATTCTGTGTAGCAGCGGTACTACTGGACTCCCAAAGGGAGTCATGCAGACCCATCAAAACATTTGCGTGCGTCTGATCCATGCTCTCGATCCACGCTACGGCACTCAGCTGATTCCTGGTGTCACCGTCTTGGTCTACTTGCCTTTCTTCCATGCTTTCGGCTTTCATATTACTTTGGGTTACTTTATGGTCGGTCTCCGCGTGATTATGTTCCGCCGTTTTGATCAGGAGGCTTTCTTGAAAGCCATCCAAGATTATGAAGTCCGCAGTGTCATCAACGTGCCTAGCGTGATCCTGTTTTTGTCTAAGAGCCCACTCGTGGACAAGTACGACTTGTCTTCACTGCGTGAATTGTGTTGCGGTGCCGCTCCACTGGCTAAGGAGGTCGCTGAAGTGGCCGCCAAACGCTTGAATCTTCCAGGGATTCGTTGTGGCTTCGGCCTCACCGAATCTACCAGTGCGATTATCCAGACTCTCGGGGATGAGTTTAAGAGCGGCTCTTTGGGCCGTGTCACTCCACTCATGGCTGCTAAGATCGCTGATCGCGAAACTGGTAAGGCTTTGGGCCCGAACCAAGTGGGCGAGCTGTGTATCAAAGGCCCTATGGTGAGCAAGGGTTATGTCAATAACGTTGAAGCTACCAAGGAGGCCATCGACGACGACGGCTGGTTGCATTCTGGTGATTTTGGATATTACGACGAAGATGAGCATTTTTACGTCGTGGATCGTTACAAGGAGCTGATCAAATACAAGGGTAGCCAGGTTGCTCCAGCTGAGTTGGAGGAGATTCTGTTGAAAAATCCATGCATTCGCGATGTCGCTGTGGTCGGCATTCCTGATCTGGAGGCCGGCGAACTGCCTTCTGCTTTCGTTGTCAAGCAGCCTGGTACAGAAATTACCGCCAAAGAAGTGTATGATTACCTGGCTGAACGTGTGAGCCATACTAAGTACTTGCGTGGCGGCGTGCGTTTTGTTGACTCCATCCCTCGTAACGTAACAGGCAAAATTACCCGCAAGGAGCTGTTGAAACAATTGTTGGTGAAGGCCGGCGGGAATTCTCACGGCTTCCCTCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGACACCCTGCTGCTTGCGCCAGC GCCAGGATCAACGTCTAA

[0179] CBRluc-hCL1-hPEST-UTR (SEQ ID NO:78)ATGGTAAAGCGTGAGAAAAATGTCATCTATGGCCCTGAGCCTCTCCATCCTTTGGAGGATTTGACTGCCGGCGAAATGCTGTTTCGTGCTCTCCGCAAGCACTCTCATTTGCCTCAAGCCTTGGTCGATGTGGTCGGCGATGAATCTTTGAGCTACAAGGAGTTTTTTGAGGCAACCGTCTTGCTGGCTCAGTCCCTCCACAATTGTGGCTACAAGATGAACGACGTCGTTAGTATCTGTGCTGAAAACAATACCCGTTTCTTCATTCCAGTCATCGCCGCATGGTATATCGGTATGATCGTGGCTCCAGTCAACGAGAGCTACATTCCCGACGAACTGTGTAAAGTCATGGGTATCTCTAAGCCACAGATTGTCTTCACCACTAAGAATATTCTGAACAAAGTCCTGGAAGTCCAAAGCCGCACCAACTTTATTAAGCGTATCATCATCTTGGACACTGTGGAGAATATTCACGGTTGCGAATCTTTGCCTAATTTCATCTCTCGCTATTCAGACGGCAACATCGCAAACTTTAAACCACTCCACTTCGACCCTGTGGAACAAGTTGCAGCCATTCTGTGTAGCAGCGGTACTACTGGACTCCCAAAGGGAGTCATGCAGACCCATCAAAACATTTGCGTGCGTCTGATCCATGCTCTCGATCCACGCTACGGCACTCAGCTGATTCCTGGTGTCACCGTCTTGGTCTACTTGCCTTTCTTCCATGCTTTCGGCTTTCATATTACTTTGGGTTACTTTATGGTCGGTCTCCGCGTGATTATGTTCCGCCGTTTTGATCAGGAGGCTTTCTTGAAAGCCATCCAAGATTATGAAGTCCGCAGTGTCATCAACGTGCCTAGCGTGATCCTGTTTTTGTCTAAGAGCCCACTCGTGGACAAGTACGACTTGTCTTCACTGCGTGAATTGTGTTGCGGTGCCGCTCCACTGGCTAAGGAGGTCGCTGAAGTGGCCGCCAAACGCTTGAATCTTCCAGGGATTCGTTGTGGCTTCGGCCTCACCGAATCTACCAGTGCGATTATCCAGACTCTCGGGGATGAGTTTAAGAGCGGCTCTTTGGGCCGTGTCACTCCACTCATGGCTGCTAAGATCGCTGATCGCGAAACTGGTAAGGCTTTGGGCCCGAACCAAGTGGGCGAGCTGTGTATCAAAGGCCCTATGGTGAGCAAGGGTTATGTCAATAACGTTGAAGCTACCAAGGAGGCCATCGACGACGACGGCTGGTTGCATTCTGGTGATTTTGGATATTACGACGAAGATGAGCATTTTTACGTCGTGGATCGTTACAAGGAGCTGATCAAATACAAGGGTAGCCAGGTTGCTCCAGCTGAGTTGGAGGAGATTCTGTTGAAAAATCCATGCATTCGCGATGTCGCTGTGGTCGGCATTCCTGATCTGGAGGCCGGCGAACTGCCTTCTGCTTTCGTTGTCAAGCAGCCTGGTACAGAAATTACCGCCAAAGAAGTGTATGATTACCTGGCTGAACGTGTGAGCCATACTAAGTACTTGCGTGGCGGCGTGCGTTTTGTTGACTCCATCCCTCGTAACGTAACAGGCAAAATTACCCGCAAGGAGCTGTTGAAACAATTGTTGGTGAAGGCCGGCGGGAATTCTGCTTGCAAGAACTGGTTCAGTAGCTTAAGCCACTTTGTGATCCACCTTAACAGCCACGGCTTCCCTCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGACACCCTGCTGCTTGCGCCAGCGCCAGGATCAACGTCTAGGGCGCGGACTTTATTTATTTATTTCTT

[0180] CBG99luc-hPEST (SEQ ID NO:79)ATGGTGAAGCGTGAGAAAAATGTCATCTATGGCCCTGAGCCTCTCCATCCTTTGGAGGATTTGACTGCCGGCGAAATGCTGTTTCGTGCTCTCCGCAAGCACTCTCATTTGCCTCAAGCCTTGGTCGATGTGGTCGGCGATGAATCTTTGAGCTACAAGGAGTTTTTTGAGGCAACCGTCTTGCTGGCTCAGTCCCTCCACAATTGTGGCTACAAGATGAACGACGTCGTTAGTATCTGTGCTGAAAACAATACCCGTTTCTTCATTCCAGTCATCGCCGCATGGTATATCGGTATGATCGTGGCTCCAGTCAACGAGAGCTACATTCCCGACGAACTGTGTAAAGTCATGGGTATCTCTAAGCCACAGATTGTCTTCACCACTAAGAATATTCTGAACAAAGTCCTGGAAGTCCAAAGCCGCACCAACTTTATTAAGCGTATCATCATCTTGGACACTGTGGAGAATATTCACGGTTGCGAATCTTTGCCTAATTTCATCTCTCGCTATTCAGACGGCAACATCGCAAACTTTAAACCACTCCACTTCGACCCTGTGGAACAAGTTGCAGCCATTCTGTGTAGCAGCGGTACTACTGGACTCCCAAAGGGAGTCATGCAGACCCATCAAAACATTTGCGTGCGTCTGATCCATGCTCTCGATCCACGCGTGGGCACTCAGCTGATTCCTGGTGTCACCGTCTTGGTCTACTTGCCTTTCTTCCATGCTTTCGGCTTTAGCATTACTTTGGGTTACTTTATGGTCGGTCTCCGCGTGATTATGTTCCGCCGTTTTGATCAGGAGGCTTTCTTGAAAGCCATCCAAGATTATGAAGTCCGCAGTGTCATCAACGTGCCTAGCGTGATCCTGTTTTTGTCTAAGAGCCCACTCGTGGACAAGTACGACTTGTCTTCACTGCGTGAATTGTGTTGCGGTGCCGCTCCACTGGCTAAGGAGGTCGCTGAAGTGGCCGCCAAACGCTTGAATCTTCCAGGGATTCGTTGTGGCTTCGGCCTCACCGAATCTACCAGCGCTAACATTCACTCTCTCGGGGATGAGTTTAAGAGCGGCTCTTTGGGCCGTGTCACTCCACTCATGGCTGCTAAGATCGCTGATCGCGAAACTGGTAAGGCTTTGGGCCCGAACCAAGTGGGCGAGCTGTGTATCAAAGGCCCTATGGTGAGCAAGGGTTATGTCAATAACGTTGAAGCTACCAAGGAGGCCATCGACGACGACGGCTGGTTGCATTCTGGTGATTTTGGATATTACGACGAAGATGAGCATTTTTACGTCGTGGATCGTTACAAGGAGCTGATCAAATACAAGGGTAGCCAGGTTGCTCCAGCTGAGTTGGAGGAGATTCTGTTGAAAAATCCATGCATTCGCGATGTCGCTGTGGTCGGCATTCCTGATCTGGAGGCCGGCGAACTGCCTTCTGCTTTCGTTGTCAAGCAGCCTGGTAAAGAAATTACCGCCAAAGAAGTGTATGATTACCTGGCTGAACGTGTGAGCCATACTAAGTACTTGCGTGGCGGCGTGCGTTTTGTTGACTCCATCCCTCGTAACGTAACAGGCAAAATTACCCGCAAGGAGCTGTTGAAACAATTGTTGGAGAAGGCCGGCGGGAATTCTCACGGCTTCCCTCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGACACCCTGCTGCTTGCGCCAGC GCCAGGATCAACGTCTAA

[0181] CBG99luc-hCL1-hPEST-UTR (SEQ ID NO:80)ATGGTGAAGCGTGAGAAAAATGTCATCTATGGCCCTGAGCCTCTCCATCCTTTGGAGGATTTGACTGCCGGCGAAATGCTGTTTCGTGCTCTCCGCAAGCACTCTCATTTGCCTCAAGCCTTGGTCGATGTGGTCGGCGATGAATCTTTGAGCTACAAGGAGTTTTTTGAGGCAACCGTCTTGCTGGCTCAGTCCCTCCACAATTGTGGCTACAAGATGAACGACGTCGTTAGTATCTGTGCTGAAAACAATACCCGTTTCTTCATTCCAGTCATCGCCGCATGGTATATCGGTATGATCGTGGCTCCAGTCAACGAGAGCTACATTCCCGACGAACTGTGTAAAGTCATGGGTATCTCTAAGCCACAGATTGTCTTCACCACTAAGAATATTCTGAACAAAGTCCTGGAAGTCCAAAGCCGCACCAACTTTATTAAGCGTATCATCATCTTGGACACTGTGGAGAATATTCACGGTTGCGAATCTTTGCCTAATTTCATCTCTCGCTATTCAGACGGCAACATCGCAAACTTTAAACCACTCCACTTCGACCCTGTGGAACAAGTTGCAGCCATTCTGTGTAGCAGCGGTACTACTGGACTCCCAAAGGGAGTCATGCAGACCCATCAAAACATTTGCGTGCGTCTGATCCATGCTCTCGATCCACGCGTGGGCACTCAGCTGATTCCTGGTGTCACCGTCTTGGTCTACTTGCCTTTCTTCCATGCTTTCGGCTTTAGCATTACTTTGGGTTACTTTATGGTCGGTCTCCGCGTGATTATGTTCCGCCGTTTTGATCAGGAGGCTTTCTTGAAAGCCATCCAAGATTATGAAGTCCGCAGTGTCATCAACGTGCCTAGCGTGATCCTGTTTTTGTCTAAGAGCCCACTCGTGGACAAGTACGACTTGTCTTCACTGCGTGAATTGTGTTGCGGTGCCGCTCCACTGGCTAAGGAGGTCGCTGAAGTGGCCGCCAAACGCTTGAATCTTCCAGGGATTCGTTGTGGCTTCGGCCTCACCGAATCTACCAGCGCTAACATTCACTCTCTCGGGGATGAGTTTAAGAGCGGCTCTTTGGGCCGTGTCACTCCACTCATGGCTGCTAAGATCGCTGATCGCGAAACTGGTAAGGCTTTGGGCCCGAACCAAGTGGGCGAGCTGTGTATCAAAGGCCCTATGGTGAGCAAGGGTTATGTCAATAACGTTGAAGCTACCAAGGAGGCCATCGACGACGACGGCTGGTTGCATTCTGGTGATTTTGGATATTACGACGAAGATGAGCATTTTTACGTCGTGGATCGTTACAAGGAGCTGATCAAATACAAGGGTAGCCAGGTTGCTCCAGCTGAGTTGGAGGAGATTCTGTTGAAAAATCCATGCATTCGCGATGTCGCTGTGGTCGGCATTCCTGATCTGGAGGCCGGCGAACTGCCTTCTGCTTTCGTTGTCAAGCAGCCTGGTAAAGAAATTACCGCCAAAGAAGTGTATGATTACCTGGCTGAACGTGTGAGCCATACTAAGTACTTGCGTGGCGGCGTGCGTTTTGTTGACTCCATCCCTCGTAACGTAACAGGCAAAATTACCCGCAAGGAGCTGTTGAAACAATTGTTGGAGAAGGCCGGCGGGAATTCTGCTTGCAAGAACTGGTTCAGTAGCTTAAGCCACTTTGTGATCCACCTTAACAGCCACGGCTTCCCTCCCGAGGTGGAGGAGCAGGCCGCCGGCACCCTGCCCATGAGCTGCGCCCAGGAGAGCGGCATGGATAGACACCCTGCTGCTTGCGCCAGCGCCAGGATCAACGTCTAGGGCGCGGACTTTATTTATTTATTTCTT

[0182] TABLE 1 Name of the primer Sequence of the primer LucN5′-AGATCTGCGATCTAAGTAAGC SEQ ID NO: 16 TTGG; LucC5′-ACTCTAGAATTCACGGCGATC SEQ ID NO: 17 TTTCC; HRLN5′-GGCGAAGCTTGGGTCACCTCC SEQ ID NO: 18 AAGGTGTACGACCCCGAGC; HRLC5′-GCTCTAGAATGAATTCTGCTC SEQ ID NO: 19 GTTCTTCAGCACGCGCT; PEST-5′5′-AATTCTCATGGCTTCCCGCCG SEQ ID NO: 8 GAGATGGAGGAGCAGGCTGCTGGCACGCTGCCCATGTCTT; PEST-3′ 5′-GTGCCCAGGAGAGCGGGATGG SEQ ID NO:9ACCGTCACCCTGCAGCCTGTGCTT CTGCTAGGATCAATGTGTAA; 5′-PEST5′-GGCCTTACACATTGATCCTAG SEQ ID NO: 10 CAGAAGCACAGGCTGCAGGGTGACGGTCCATCCCGCTCTCCT; 3′-PEST 5′-GGGCACAAGACATGGGCAGCG SEQ ID NO: 11TGCCAGCAGCCTGCTCCTCCATCT CCGGCGGGAAGCCATGAG; Ubiquitin5′-TAGCATGGTCACCCAGATTTT SEQ ID NO: 20 5′wt/BsytEII CGTGAAAACCCTTACG;Ubiquitin 5′-ATGCTAGGTGACCGGATCCCG SEQ ID NO: 21 3′w/BstEIICGGATAACCACCA; 5′-CCATGGGACATCATCACCATC SEQ ID NO: 22ACCACGGGGATCCACAAGCTTATG AAGAAATTAGCAA; Ubi-Luc 3′5′-TTCTGGATCCCGCGGTATACC SEQ ID NO: 23 w/Linker ACCACGAAGACTCAACAC;Ubi-Luc 3′ 5′-TTCTGGATCCCGCGGCATACC SEQ ID NO: 24 w/Linker MetACCACGAAGACTCAACAC; Ubi-Luc3′ 5′-TTCTGGATCCCGCGGCTCACC SEQ ID NO: 25w/Linker Glu ACCACGAAGACTCAACAC; Ubi-Luc 5′ 5′-TATGGGCCCTTAATACGACTC SEQID NO: 26 w/Linker ACTATAGGGGAATTGTGAGCGGAT AACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATA TAC CATGCAGATTTTCGTGAAAA CC; Ubiq(R)5′-TTTTGGCGTCGGTGACCGGAT SEQ ID NO: 27 CCCGCGGTCGACCACCACGAAG; Ala5′-TTTTGGCGTCGGTGACCGGAT SEQ ID NO: 28 CCCGCGGTGCACCACCACGAAG; Asn5′-TTTTGGCGTCGGTGACCGGAT SEQ ID NO: 29 CCCGCGGGTTACCACCACGAAG; Asp5′-TTTTGGCGTCGGTGACCGGAT SEQ ID NO: 30 CCCGCGGATCACCACCACGAAG; Phe5′-TTTTGGCGTCGGTGACCGGAT SEQ ID NO: 31 CCCGCGGGAAACCACCACGAAG; His25′-TTTTGGCGTCGGTGACCGGAT SEQ ID NO: 32 CCCGCGGATGACCACCACGAAG; His5′-TTTTGGCGTCGGTGACCGGAT SEQ ID NO: 33 CCCGCGGGTGACCACCACGAAG; Leu5′-TTTTGGCGTCGGTGACCGGAT SEQ ID NO: 34 CCCGCGGGAGACCACCACGAAG; Lys5′-TTTTGGCGTCGGTGACCGGAT SEQ ID NO: 35 CCCGCGGCTTACCACCACGAAG; Gln5′-TTTTGGCGTCGGTGACCGGAT SEQ ID NO: 36 CCCGCGGTTGACCACCACGAAG; Trp5′-TTTTGGCGTCGGTGACCGGAT SEQ ID NO: 37 CCCGCGGCCAACCACCACGAAG;Ubiq(E)del5′ 5′-GTTTTTGGCGTCGGTGACCTC SEQ ID NO: 38 ACCACCACGAAGACTC;Ubiq(E)del3′ 5′-GAGTCTTCGTGGTGGTGAGGT SEQ ID NO: 39 CACCGACGCCAAAAAC;Luc 5′ 5′-GTTCCAGGAACCAGGGCGTAT SEQ ID NO: 40 CTC; Luc 3′5′-CGCGGAGGAGTTGTGTTTGTG SEQ ID NO: 41 GAC; Luc + N5′-GGCGAAGCTTGGGTCACCGAT SEQ ID NO: 42 GCTAAGAACATTAAGAAGGG; Luc + C5′-GCTCTAGAATGAATTCACGGC SEQ ID NO: 43 GATCTTGCCGCC; tetO-5′5′-GATTAATGGCCCTTTCGTCCT SEQ ID NO: 50 TCGAGTT; tetO-3′5′-AGCTAGCGAGGCTGGATCGGT SEQ ID NO: 51 CCCGGT; AUUU5′-CTAGATTTATTTATTTATTTC SEQ ID NO: 52 TTCATATGC; Anti-AUUU5′-AATTGCATATGAAGAAATAAA SEQ ID NO: 53 TAAATAAAT; hsp70-5′5′-ATTAATCTGATCAATAAAGGG SEQ ID NO: 54 TTTAAGG; hsp70-3′5′-AAAAAGGTAGTGGACTGTCG; SEQ ID NO: 55 3′-BKB1 5′-AATTGGGAATTAAAACAGCATSEQ ID NO: 56 TGAACCAAGAAGCTTGGCTTTCTT ATCAATTCTTTGTGACATAATAAG TT;5′-BKB1rev 5′-AACTTATTATGTCACAAAGAA SEQ ID NO: 57TTGATAAGAAAGCCAAGCTTCTTG GTTCAATGCTGTTTTAATTCCC; N—M5′-GATCTGCGGCCGCATATATG; SEQ ID NO: 58 M—C 5′-GTGACCATATATGCGGCCGC SEQID NO: 59 A; CL1-RI-final 5′-AATTTGTCTGCCTGCAAGAAC SEQ ID NO: 60TGGTTCAGCAGCTTGAGCCACTTC GTGATCCACTTG; Rev-CL1-RI-5′-AATTCAAGTGGATCACGAAGT SEQ ID NO: 61 final GGCTCAAGCTGCTGAACCAGTTCTTGCAGGCAGACA; CL1 5′-AATTCTGCCTGCAAGAACTGG SEQ ID NO: 62TTCAGCAGCTTGAGCCACTTCGTG ATCCACTTGTAAGC; Rev-CL15′-GGCCGCTTACAAGTGGATCAC SEQ ID NO: 63 GAAGTGGCTCAAGCTGCTGAACCAGTTCTTGCAGGCAG; CL1-N 5′-GATCTTATGTCTGCCTGCAAG SEQ ID NO: 64AACTGGTTCAGCAGCTTGAGCCAC TTCGTGATCCACTTGCA; Rev-CL1-N5′-AGCTTGCAAGTGGATCACGAA SEQ ID NO: 65 GTGGCTCAAGCTGCTGAACCAGTTCTTGCAGGCAGACATAA; AUUU 5′-CTAGATTTATTTATTTATTTC SEQ ID NO: 3 TTCATATGC;Anti-AUUU 5′-AATTGCATATGAAGAAATAAA SEQ ID NO: 4 TAAATAAAT;

[0183] DNA Sequencing

[0184] Plasmid DNA sequences were confirmed by DNA sequencing which wasperformed on ABI Prizm Model 377 using either Luc5′ or Luc3′ primers(Table 1).

[0185] Expression In Vitro

[0186] Both TNT® SP6 Coupled Wheat Germ Extract System and TNT® T7Coupled Reticulocyte Lysate System (Promega) were used to expressfirefly luciferase and fusion proteins thereof in vitro. [³H]-Leucinewas included in the reaction mixture. Upon completion, the reactionmixtures were separated into two portions. The first portion was used todetermine luciferase activity as described in the section entitled“Luciferase assay conditions” and the second portion was used todetermine the quantity of synthesized luciferase. Proteins contained inthe second portion were separated by SDS gel electrophoresis using 4-20%Tris-glycine gels (Novex). After completion of the electrophoresis, thelocation of the protein of interest on the gel was determined byautoradiography. Then bands containing proteins of interest were cutfrom the gel and the amounts of incorporated radioactivity determined byliquid scintillation. The ratio between luminescence data and the amountof radioactivity was used to characterize specific activity.

[0187] Mammalian Cells

[0188] Human adenocarcinoma cell line HeLa, African green monkey kidneycell line COS-7, Chinese hamster ovary cell line CHO-K1, and humanembryonic kidney 293 cells were obtained from ATCC. All cell lines weremaintained in RPMI-1640 medium containing 5% fetal bovine serum and amixture of antibiotics (penicillin, 100 μg/ml; streptomycin, 100 μg/ml;amphotericin B, 0.25 g/ml).

[0189] Transient Transfection and Treatment with Cycloheximide

[0190] For transfection, cells were grown to confluence in T25 flasks(Falkon, Becton Dickinson, Oxford). Transfection was conducted in 1 mlof serum-free RPMI-1640 that was mixed with 8 μg of plasmid DNA and 20μl of Lipofectamine™2000 (GIBCO BRL). CHO cells were incubated in thetransfection media for 30 minutes, HeLa cells for 1 hour, and COS-7cells for 5 hours. Following incubation, cells were trypsinized withTrypsin-EDTA (GIBCO BRL) and collected by centrifugation. Cellscollected from each T25 flask were resuspended in 10 ml of RPMI-1640medium containing 5% fetal bovine serum and a mixture of antibiotics,transferred to 96 well plates (100 μl/well), and allowed to grow for12-16 hours prior to treatment with different agents. Cycloheximide(Sigma) or doxycycline was added to different wells at different times(the final concentration was 100 μg/ml and 2 μg/ml, respectively). Forthermoinduction, cells were transferred to 42° C. for 1 hour and thenwere incubated at 37° C. for different periods. After incubation, plateswith cells expressing derivatives of firefly luciferase were transferredto −70° C. In the case of cells expressing Renilla luciferase, theculture media was substituted with lysis buffer from the RenillaLuciferase Assay System (Promega).

[0191] Luciferase Assay Conditions

[0192] To determine firefly luciferase activity, cells were lysed byfreeze/thawing. Lysate from each well (100 μl) was transferred intocorresponding wells of opaque 96 well plates (Falkon, Becton Dickinson,Oxford) containing 100 μl of Bright-Glo™ Luciferase Assay System(Promega) and luminescence intensity was determined using MLX MicrotiterPlate Luminometer (Dynex). The activity of Renilla luciferase wasmeasured by mixing 40 μl samples with 100 μl of assay reagent from theRenilla Luciferase Assay System (Promega).

[0193] Tet-Off System

[0194] A DNA fragment containing the CMV minimal promotor withheptamerized upstream tet-operators (Gossen et al., 1992) was amplifiedfrom plasmid pUHD10-3 by PCR with primers: AGCTAGCGAGGCTGGATCGGTCCCGGT(SEQ ID NO:44) and GATTAATGGCCCTTTCGTCCTCGAGTT (SEQ ID NO:45). Theamplified fragment was used to substitute the CMV promoter into Renillaluciferase encoding plasmids. HeLa cells were transfected with a mixturecontaining a Renilla luciferase encoding plasmid and plasmid pUHD 15-1in ratio of 4.5:1. Plasmid pUHD 15-1 encodes a hybrid transactivatorthat contains the tetracycline repressor and the C-terminal domain ofVP16 from HSV which stimulates minimal promoters fused to tetracyclineoperator sequences. In the presence of doxycycline, activity of thehybrid transactivator is inhibited.

[0195] CRE System

[0196] D293 cells are an isolated subpopulation of 293 cells thatproduce a significant amount of cAMP upon induction. pGL-3 plasmidscontain multiple CREs which respond to cAMP induction by increasingtranscription. D293 cells were trypsin treated and 7.5×10³ cells wereadded to wells of a 96-well plate. After an overnight incubation, themedia from transfected cells was removed and replaced with mediacontaining isoproterenol (Iso, Calbiochem #420355, final concentration 1μM) and RO (Ro-20-1724, Calbiochem #557502, final concentration 100 μM).Iso induces the cAMP pathway and RO prevents degradation of cAMP. Theplates were returned to the incubator. Samples from time points t=0, 3,6, 9 and 12 hours post-Iso/RO were collected. At 6 or 24 hourspost-Iso/RO addition, prostaglandin E1 (PGE1, Calbiochem #538903, finalconcentration 1.0 μM) was added to the cells. Samples from time points24, 27, 30, 33, and 36 hours post-Iso/RO addition (i.e., experimentalstart) were then collected.

[0197] For click beetle experiments, D293 cells were transientlytransfected with codon optimized red (CBR) or green (CBG) click beetlesequences in conjunction with destabilization sequences. Two dayspost-transfection, the cells were treated with trypsin and 7.5×10³ cellswere added to wells of a 96-well plate. After an overnight incubation,the media from transfected cells was removed and replaced with mediacontaining isoproterenol. Samples from time points t=0, 1.5, 3, 4.5, 6,and 7.5 hours post-induction were collected.

[0198] To determine the relative light units (RLU) for the samples, 20μl of passive lysis buffer was added to each well, the Dual LuciferaseAssay was performed, and the RLU were collected. In order to determinethe fold-induction, the RLU from the drug treated wells were divided byRLU from non-treated wells.

[0199] Stable Cell Lines

[0200] D293 cells were transfected with plasmids and then grown in mediacontaining 600 μg/ml of G418. Individual lines of stably transfectedcells were generated by seeding individual cells from the populationgrown in the G418-containing media into wells of a 96-well plate andgrowing the seeded cells in the G418-containing media.

[0201] Results

[0202] Construction and Analysis of Deubiqutination of Ubiquitin-FireflyLuciferase Fusions

[0203] To create luciferase species that would have at their N-terminiamino acid residues of choice, the approach described earlier byVarshavsky and coauthors was used (Bachmair et al., 1986). This approachutilizes the ability of ubiquitin-specific processing proteases in cellsto cleave fusion proteins containing ubiquitin at the N-terminus. Suchcleavage occurs immediately after the last amino acid residue ofubiquitin. According to Bachlmair et al. (1986) such deubiquitinationoccurs irrespective of the identity of the residue located immediatelyafter the cleavage site.

[0204] Plasmids pUbiq(Y)Luc 19 and pSPUbiqLuc1 encode ubiquitin-fireflyluciferase fusion proteins containing a tyrosine residue immediatelyafter the ubiquitin sequence. Plasmid pUbiq(Y)Lucl9 was designed to beexpressed in mammalian cells and possesses an early promoter of CMVupstream and an SV40 polyadenylation signal downstream of the sequenceencoding the fusion protein. Plasmid pSPUbiqLucl encodes the sameprotein as pUbiq(Y)Lucl9 but possesses a promoter recognized by the DNApolymerase of bacteriophage SP6. Therefore, plasmid pSPUbiqLuc1 can beused for in vitro production of mRNA encoding the fusion protein. Bothof these plasmids were used to confirm that in eukaryotic cells, and inmammalian cells specifically, ubiquitin-firefly luciferase fusionproteins undergo deubiquitination.

[0205] As shown in FIG. 1, both in a wheat germ based in vitrotranslation system as well as in transiently transfected mammaliancells, expression of recombinant genes encoding ubiquitin-fireflyluciferase fusion proteins resulted in accumulation of proteins that hadmolecular masses expected for wild-type firefly luciferase. The controlwild-type firefly luciferase in these experiments was encoded either byplasmid pETUbiqLuc or pwtLuc1. In addition to the major band, a minorband was also present on the autoradiograph of proteins generated in thewheat germ based system. This minor band corresponds to the full-sizerecombinant protein that was not deubiquitinated. In several experimentsperformed using CHO cells, the minor band was either much weaker than inthe wheat germ based system or was not detectable at all, suggestingthat within mammalian cells, deubiquitination of luciferase happens veryquickly and efficiently.

[0206] Comparison of Specific Activities of Wild Type Luciferase and itsDerivatives

[0207] Sung et al. (1995) reported that modifications of the fireflyluciferase N-terminal region could interfere with enzymatic activity.Thus, luciferase species generated as a result of deubiquitinationprocess might have different enzymatic activities than that of wild-typeluciferase. To assess specific activities of luciferase fusion proteinsand compare them with the specific activity of wild-type luciferase,plasmids pT7Ubiq(Y)Luc19.2 and pT7 Ubiq(E)Luc19.1 were constructed sothat, in addition to an eukaryotic promoter, they have a promoter ofbacteriophage T7. As a result, these plasmids direct the synthesis ofluciferase fusion proteins in an in vitro transcription/translationsystem. Plasmid pT7Ubiq(Y)Luc 19.2 encodes exactly the same protein asthat encoded by plasmid pUbiq(Y)Luc19. Plasmid pT7 Ubiq(E)Luc19.1encodes a ubiquitin-firefly luciferase fusion protein that differs fromthe protein encoded by plasmid pT7Ubiq(Y)Luc19.2 in only one position.In this position, located immediately after the last amino acid residueof ubiquitin, the protein encoded by plasmid pT7Ubiq(E)Luc19.1 has aglutamic acid residue in place of a tyrosine residue. Additionally,plasmids pETwtLucl and pT7Luc-PEST10 were constructed that have apromoter of bacteriophage T7 and encode wild-type firefly luciferase ora fusion protein comprising firefly luciferase and a mutant form ofC-ODC, respectively.

[0208] Plasmids encoding wild-type luciferase as well as a luciferasefusion protein were used in a rabbit reticulocyte in vitrotranscription/translation system to determine luciferase activitiesaccumulated in each reaction mixture and normalize these activities bythe amount of radioactive leucine incorporated in correspondingluciferase species. Data presented in FIG. 1 (panel C) demonstrate that,similarly to that found in CHO cells, only deubiquitinated forms ofluciferase were accumulated in rabbit reticulocyte in vitrotranscription/translation systems supplemented with either plasmidpT7Ubiq(Y)Luc19.2 or pT7Ubiq(E)Luc19.1. The presence in these mixturesof small proteins with electrophoretic mobilities of free ubiquitinevidences that full-size ubiquitin-luciferase fusions were produced inthese reactions and were efficiently deubiquitinated. On the contrary,in the case of plasmid pT7Luc-PEST10, accumulation of the full-sizeprotein was observed. Nevertheless, the data presented in FIG. 2demonstrate that both deubiquitinated forms of luciferase haveessentially the same specific activity as that of wild-type luciferaseand the protein encoded by plasmid pT7Luc-PEST10.

[0209] Comparison of Efficiencies of Luciferase Destabilization byN-Degron and a Mutant of the C-Terminus of m-ODC

[0210] The half-life of the protein encoded by plasmid pUbiq(Y)Luc 19was determined in mammalian cells and compared to the half-lives ofwild-type luciferase as well as fusion proteins comprising fireflyluciferase and a mutant form of the C-ODC (Luc-PEST10). This was done byevaluating the luminescence emitted by cells that were transientlytransfected with either plasmid pUbiq(Y)Luc19 or pwtLuc1 or pLuc-PEST10, respectively, and then, for different periods of time, were exposedto the protein synthesis inhibitor cycloheximide. In this experiment,cycloheximide was used at a concentration that caused completeinhibition of protein synthesis within 7.5 minutes of exposure (100μg/ml). Results presented in FIG. 3 demonstrate that in COS-7 cells, theUbiq(Y)Luc fusion protein has an intermediate half-life compared to thatof wild-type luciferase and a Luc-PEST fusion protein. The estimatedhalf-life of Ubiq(Y)Luc in these cells is about 170-200 minutes, whichis far longer than the half-lives reported by Varshavsky (1992) forP-galactosidase containing a tyrosine residue at the N-terminus both inE. coli and S. cerevisiae (2 and 10 minutes, respectively). At the sametime, the half-life of wild-type luciferase in COS-7 cells (about 7hours) was shorter than the half-life of the wild-type P-galactosidaseboth in E. coli and in S. cerevisiae (more than 10 and 20 hours,respectively), suggesting that in COS-7 cells, N-degron initiateddegradation occurs less efficiently than in yeast and bacteria. The factthat the half-life of Luc-PEST in these cells (about 120 minutes) isshorter than the half-life of Ubiq(Y)Luc suggests that the proteindegrading capacity of the proteosome is not a limiting parameter fordetermining the half-life of Ubiq(Y)Luc. When the same analysis wasperformed using either CHO or HeLa cells, it was found that degradationof all three proteins in these cells occurs slightly faster than inCOS-7 cells (see FIG. 7).

[0211] Analysis of the N-End Rule In Different Cell Lines

[0212] To understand to what degree in mammalian cells the N-end rulemight differ from the same rule in yeast and E. coli, a set of plasmidswas created that encode an ubiquitin-firefly luciferase fusion protein,namely pUbiq(Y)Lucl9, pT7Ubiq(Y)Luc19.2 and pT7Ubiq(E)Luc19.1 (for alist of plasmids see Table 2). Proteins encoded by these plasmids haveonly one difference in their sequences; the amino acid residue locatedimmediately after the ubiquitin sequence. Therefore, upondeubiquitination, these proteins should generate firefly luciferasespecies that are different only in the first N-terminal amino acidresidue. Plasmids by themselves have additional differences in theregion located upstream of the fusion protein coding region. Some ofthese plasmids have an additional bacteriophage T7 promoter (plasmidsdesignated pT7Ubiq(X)Luc). Nevertheless, as shown in FIG. 3 (see curvesfor plasmids pT7Ubiq(Y)Luc19.2 and pUbiq(Y)Luc19), the presence or theabsence of bacteriophage T7 promoter had no effect on the stability ofcorresponding proteins. TABLE 2 Amino acid residue following immediatelyPlasmid after ubiquitin sequence PT7Ubiq(M)Luc19.2 Met PT7Ubiq(E)Luc19.1Glu PT7Ubiq(I)Luc19.1 Ile pUbiq(R)Luc13 Arg pUbiq(F)Luc10 PhepUbiq(A)Luc2 Ala pUbiq(N)Luc25 Asn pUbiq(W)Luc16 Trp pUbiq(L)Luc23 LeupUbiq(Q)Luc36 Gln pUbiq(Y)Luc19 Tyr pUbiq(Asp3)Luc16 Asp pUbiq(His2)Luc3His pUbiq(K)Luc4 Lys

[0213] The stabilities of proteins encoded by plasmids listed in Table 2were analyzed using cell lines derived from three different species,hamster (CHO), monkey (COS-7) and human (HeLa). It was observed thatwith one exception, Ubiq(M)Luc in HeLa cells, all ubiquitin containingfirefly luciferase fusion proteins were degraded faster than wild-typeluciferase (FIG. 4). Patterns of relationships between amino acidresidues positioned at the N-terminus of luciferase and the half-life ofthe corresponding protein remained essentially unchanged from experimentto experiment. For all three cell lines, a glutamic acid residue wasfound to be the most efficient destabilizing residue among all residuestested. Aspartic acid was also found to be a quite efficientdestabilizing residue. At the same time, in all three cell lines, basicamino acid residues were found to have relatively weak destabilizingproperties. In CHO cells, the difference between the half-lives of themost stable and the least stable constructs was almost two times smallerthan the same differences determined in COS-7 and HeLa cells. Thus, indifferent cell lines, the N-end rule might have a different role indetermining the fate of the protein.

[0214] In general, a correlation was observed between the half-life ofthe specific protein and the luminescence of cells producing thisprotein. Indeed, in FIG. 4, most of the symbols characterizing therelationship between luminescence intensities and half-lives of fireflyluciferase fusion protein tend to locate along straight lines.Nevertheless, in some cases such a correlation was not observed. Forexample, in COS-7 cells, the protein encoded by plasmid pUbiq(A)Luc2produced much less luminescence than would be expected based on itshalf-life in these cells. Moreover, when the same protein was producedin CHO or HeLa cells, a dramatic deviation from the general rule was notobserved. The potential role of inconsistencies in transfectionefficiency in generating this phenomena was addressed by introducing, inaddition to the plasmid encoding the firefly luciferase fusion protein,the same amount of plasmid encoding Renilla luciferase to eachtransfection mixture. Renilla luminescence in the transfected cells wasused as a measure of transfection efficiency and to normalize fireflyluminescence produced by the same cells. Normalization did not eliminatethe observed inconsistencies, suggesting that such inconsistencies mightreflect biological characteristics of the specific protein-cell pairs.

[0215] Dependence of the N-End Rule on The Protein Structure

[0216] To understand dominance of the N-terminal residue in determiningthe fate of the protein within mammalian cells, the stabilities ofluciferase fusion proteins having the same amino acid residue at theN-terminus, but different peptide sequences attached to the fireflyluciferase, were compared. As shown in FIG. 5, proteins encoded byplasmids pUbiq(E)ΔLuc6 and pUbiq(H)ΔLuc18 have four amino acid deletionswhen compared to proteins encoded by plasmids pT7Ubiq(E)Luc19.1 andpUbiq(His2)Luc3, respectively. In the protein encoded by plasmidpUbiqLuc15, one glutamic acid residue substitutes for residues found inthe protein encoded by pUbiq(Y)Luc 19.

[0217] Data presented in FIG. 6 reveal that proteins with the sameN-terminal residue could have dramatically different half-livesdepending on the structure of the following sequence. For example, inCOS-7 cells, the half-life of the protein encoded by plasmidpUbiq(Y)Luc19 was about 140 minutes while the half-life of the proteinencoded by plasmid pUbiqLuc15 was almost 7 hours. Depending on the cellsused for the experiment, this difference could be less dramatic. Indeed,the same proteins when analyzed in CHO cells had half-lives of about 190minutes and about 240 minutes, respectively. Surprisingly, depending onthe amino acid residue located at the N-terminus, the effect of changesin the following sequence could be different. Indeed, deletion of fouramino acid residues in a protein that has a histidine residue at theN-terminus resulted in destabilization of the protein (in COS-7 cellsthe half-life was reduced from about 300 minutes to about 120 minutesand in CHO cells, from about 320 minutes to about 200 minutes). At thesame time, when the N-terminal position was occupied by an aspartic acidresidue, the same deletion resulted in stabilization of the protein inCOS-7 cells (the half-life changed from about 60 minutes to about 200minutes) and had essentially no effect on the stability of thecorresponding protein in CHO cells. Thus, even though the N-terminalresidue plays an important role in the determining protein stability, itis not the dominant factor.

[0218] Optimization of the Reporter Properties of Firefly Luciferase

[0219] It had been reported that degradation of ODC occurs in the 26Sproteosome without prior ubiquitination (Bercovich et al., 1989;Murakami et al., 1992), while degradation directed by N-degron wasreported to be a ubiquitin-dependent process. To determine whether thecombination of N-degron and C-ODC in the same protein might direct therecombinant protein towards degradation in the proteosome by twodifferent pathways, thus decreasing the half-life of the protein evenfurther, three plasmids, pUbiq(Y)Luc-PEST5, pUbiq(R)Luc-PEST12 andpT7Ubiq(E)Luc-PEST23, were constructed. Sequences of proteins encoded bythese plasmids are different only in the position that followsimmediately after the last amino acid residue of the ubiquitin sequence.In that position, the protein encoded by plasmid pUbiq(Y)Luc-PEST5 has atyrosine, the protein encoded by plasmid pUbiq(R)Luc-PEST12 has anarginine, and the protein encoded by plasmid pT7Ubiq(E)Luc-PEST23 has aglutamic acid residue. The stabilities of proteins encoded by theseplasmids were tested in HeLa (FIG. 7), COS-7 and CHO cells. The analysisrevealed that in all cell lines, proteins with two degradation signalswere less stable than proteins that contained only one signal. Forexample, in HeLa cells, ubiquitin-luciferase fusion proteins Ubiq(E)Lucor Luc-PEST 10 had half-lives of 70 and 65 minutes, respectively (FIG. 4and FIG. 7). At the same time, the half-lives of proteins encoded byplasmids pUbiq(Y)Luc-PEST5, pUbiq(R)Luc-PEST12 and pT7Ubiq(E)Luc-PEST23were 55, 40 and 30 minutes, respectively. Additionally, a combination oftwo degradation signals in the same protein resulted in more consistentdegradation from cell line to cell line. For example, when threedifferent cell lines were used to determine the half-life of the proteinUbiq(E)Luc that contains only the N-degron, the value varied from 70 to150 minutes (FIG. 4). When the same cell lines were used to determinethe half-life of the protein encoded by plasmid pT7Ubiq(E)Luc-PEST23,the half-life was 30 minutes for HeLa cells (FIG. 7), 45 minutes forCOS-7 cells and 40 minutes for CHO cells.

[0220] Luminescence data from cells transfected with plasmids encodingluciferase with another combination of protein destabilization sequencesare shown in FIG. 8. The presence of CL-1 and PEST in a luciferasefusion protein resulted in a protein that had a reduced half-liferelative to a luciferase fusion protein with either CL-1 or a PESTsequence.

[0221] To determine whether optimized codon sequences can enhance theamount of light emitted in cells transfected with a plasmid encoding aluciferase fusion protein, plasmid pT7Ubiq(E)hLuc+PEST80 wasconstructed, which encodes the same protein as that encoded by plasmidpT7Ubiq(E)Luc-PEST23, except that it contains a luciferase encodingsequence that has been optimized for expression in human cells. As aresult, translation of the fusion protein proceeds more efficiently whenthis protein is encoded by plasmid pT7Ubiq(E)hLuc+PEST80 rather than byplasmid pT7Ubiq(E)Luc-PEST23. As shown in FIG. 7, the luminescence ofcells transfected with plasmid pT7Ubiq(E)hLuc+PEST80 becomes comparableto that of cells producing wild-type firefly luciferase. Therefore,codon optimized sequences compensate for loss of expression whenincorporated with a destabilization sequence as compared to adestablilization sequence in conjunction with a non-optimized codonsequence.

[0222] To determine whether a mRNA destabilization sequence in the mRNAfor a fusion polypeptide comprising a luciferase and a proteindestabilization sequence could further decrease the half-life ofexpression of a luciferase encoded by an optimized sequence, plasmidswith promoters linked to optimized Renilla luciferase sequences andvarious combinations of destabilization sequences were tested (FIG. 9).Generally, the greater the number of destabilization sequences, theshorter the half-life of expression of the encoded protein.

[0223]FIGS. 10 and 12-16 show luminescence after the induction ofexpression of optimized Renilla, firefly or click beetle luciferasesequences from plasmids having various combinations of destabilizationsequences. Plasmids with more destabilization sequences generally hadbetter response profiles than those with no or fewer destabilizationsequences, i.e., destabilized reporters respond faster and theirrelative activation is higher than that of more stable derivatives.

[0224]FIG. 13 demonstrates that reporters can respond to two subsequentstimuli and that destabilized reporters are more suitable than stablereporters for detection of subsequent stimuli (when two stimuli occur ina relative short period of time) because a stable reporter does not havetime to react. In the bottom graph of FIG. 13, the curve correspondingto the stable version of optimized firefly luciferase continues toincrease. At the same time, the curve corresponding to the destabilizedprotein, after reaching a maximum, begins to decrease, and only afterthe addition of hCG begins to increase again.

[0225] To determine whether destabilized reporter proteins in stablytransfected cell lines could be detected, D293 cells were transfectedwith plasmids containing luciferase encoding sequences under the controlof a cAMP regulated promoter. Plasmid pCRE-hLuc+Kan18 encodes a stableversion of firefly luciferase, plasmid pCRE-hLucP+Kan8 encodes aluciferase fusion that has a PEST sequence at the C-terminus, andplasmid pCRE-hLucCP+Kan28 encodes a firefly luciferase fusionpolypeptide that has CL1-PEST sequences as well as mRNA that has a mRNAdestabilization sequence (UTR). G418-resistant clones were treated for 7hours with 10 μM of forskolin or incubated for the same period of timein forskolin-free media. After the completion of the incubation period,luminescence was determined using Bright-Glo reagent (FIG. 14). Stableclones with destabilized constructs were generally as bright as stableclones with a nondestabilized construct.

[0226] Discussion

[0227] Varshavsky and coauthors determined that both in yeast and E.coli cells there is a definite correlation between the identity of theN-terminal residue and the half-life of the corresponding protein (theN-end rule). These findings suggested that the residue located at theN-terminus of the protein might play an important role in determiningthe fate of the protein inside bacterial and yeast cells. The dataherein demonstrate that in different mammalian cells, depending on theidentity of the N-terminal residue, the half-life of firefly luciferasefusion proteins might vary from 420 to 70 minutes, suggesting that theN-end rule functions in mammalian cells. Nevertheless, the N-end rule inmammalian cells is surprisingly different from the N-end rule describedearlier for yeast and E. coli. Indeed, arginine and lysine residues wereidentified as the most destabilizing residues both in yeast and bacteria(Varshavsky, 1992). In contrast, these residues were just moderatelydestabilizing in mammalian cells. In all cell lines tested, a glutamicacid residue had the most dramatic effect on the stability of fireflyluciferase. At the same time, according to Varshavsky (1992), thisresidue in yeast had a moderate destabilizing effect and in E. coliglutamic acid had no destabilizing effect at all.

[0228] The data demonstrate that, in addition to the nature of the hostcells, the structure of the protein may have an effect on the N-endrule. Indeed, by introducing a small deletion in the area that, afterdeubiquitination, becomes the N-terminus, the relative destabilizingproperties of histidine and glutamic acid residues could be changed. Asa result, in COS-7 cells, luciferase fusion proteins with a N-terminalhistidine residue became even more unstable than the correspondingprotein with a glutamic acid residue at the N-terminus. It is importantto mention that this effect was cell-specific. In CHO cells, the samedeletion reduced the half-life of the protein possessing a histidineresidue at the N-terminus but did not change the half-life of theprotein possessing a glutamic acid residue at the N-terminus (see FIG.6) and, as a result, did not change the status of glutamic acid as amore efficient destabilizing residue than histidine.

[0229] Moreover, the addition of a mODC fragment to the C-terminus offirefly luciferase changed the half-life of corresponding proteins butat the same time did not alter the destabilizing effect of a glutamicacid residue relative to arginine and tyrosine. Further, glutamic acidappears to be the most efficient destabilizing residue in the case ofRenilla luciferase (data not presented). Therefore, the N-end ruledetermined for one protein may provide a useful guidance todestabilization of other proteins.

[0230] In mammalian cells, the N-degron dependent degradation pathwaymay function less efficiently than it does in yeast cells. Indeed, bypositioning Arg, Lys, Phe, Leu, Trp, His, Asp or Asn at the N-terminusof P-galactosidase, Varshavsky and coauthors (Bachmair et al., 1986)were able to reduce the half-life of β-galactosidase in yeast from 20hours to 2-3 minutes. At the same time, in a mammalian cell, even withglutamic acid at the N-terminus, firefly luciferase had a half-life ofgreater than 45-50 minutes. When compared to the protein degradationsignal contained within the C-ODC, N-degron alone does not provide asuperior approach to the destabilization of proteins. Nevertheless, thedata demonstrate that N-degron and C-ODC can complement each other andthe combination of these two degradation signals on the same proteinresults in an increased rate of protein degradation. Using thiscombination of degradation signals, a firefly luciferase was generatedthat in mammalian cells has the shortest half-life among currentlydescribed reporter proteins. In contrast, a protein having a degradationsignal from listeriolysin 0 and from murine C-ODC had a rate of proteindegradation which was similar to a protein having a degradation signalfrom murine C-ODC (data not shown).

[0231] The rapid turnover of reporter proteins in the cell may beinvaluable for monitoring fast processes in the cell. Additionally,destabilized reporters can allow for a substantial reduction of time inhigh-throughput screening experiments. One major disadvantage ofdestabilized reporter proteins is related to the fact that, because ofreduced quantities of such proteins in the cell, the signal availablefor detection and analysis is weaker than the signal generated bywild-type reporter proteins. For example, cells producing fireflyluciferase fusion proteins that possess both N-degron and C-ODC emitalmost ten times less light than the same cells producing wild-typeluciferase (see FIG. 7). Nevertheless, optimization of the sequenceencoding reporter protein provides a useful approach to overcome thislimitation. Indeed, by using this approach, the signal emitted by cellsproducing destabilized firefly luciferase was increased almosteight-fold without affecting the half-life of the reporter.

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[0271] All publications, patents and patent applications areincorporated herein by reference. While in the foregoing specification,this invention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details herein may be varied considerably without departing fromthe basic principles of the invention.

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FEATURE:<223> OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 9 gtgcccaggagagcgggatg gaccgtcacc ctgcagcctg tgcttctgct aggatcaatg 60 tgtaa 65 <210>SEQ ID NO 10 <211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: A synthetic primer<400> SEQUENCE: 10 ggccttacac attgatccta gcagaagcac aggctgcagggtgacggtcc atcccgctct 60 cct 63 <210> SEQ ID NO 11 <211> LENGTH: 63<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 11 gggcacaagacatgggcagc gtgccagcag cctgctcctc catctccggc gggaagccat 60 gag 63 <210>SEQ ID NO 12 <211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: A synthetic CL1sequence <400> SEQUENCE: 12 Ala Cys Lys Asn Trp Phe Ser Ser Leu Ser HisPhe Val Ile His Leu 1 5 10 15 <210> SEQ ID NO 13 <211> LENGTH: 57 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic oligonucleotide <400> SEQUENCE: 13 aattcaagtggatcacgaag tggctcaagc tgctgaacca gttcttgcag gcagaca 57 <210> SEQ ID NO14 <211> LENGTH: 57 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: A synthetic oligonucleotide<400> SEQUENCE: 14 aatttgtctg cctgcaagaa ctggttcagc agcttgagccacttcgtgat ccacttg 57 <210> SEQ ID NO 15 <211> LENGTH: 120 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic optimized PEST sequence <400> SEQUENCE: 15cacggcttcc ctcccgaggt ggaggagcag gccgccggca ccctgcccat gagctgcgcc 60caggagagcg gcatggatag acaccctgct gcttgcgcca gcgccaggat caacgtctaa 120<210> SEQ ID NO 16 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: A syntheticprimer <400> SEQUENCE: 16 agatctgcga tctaagtaag cttgg 25 <210> SEQ ID NO17 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: A synthetic primer <400>SEQUENCE: 17 actctagaat tcacggcgat ctttcc 26 <210> SEQ ID NO 18 <211>LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 18ggcgaagctt gggtcacctc caaggtgtac gaccccgagc 40 <210> SEQ ID NO 19 <211>LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 19gctctagaat gaattctgct cgttcttcag cacgcgct 38 <210> SEQ ID NO 20 <211>LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 20tagcatggtc acccagattt tcgtgaaaac ccttacg 37 <210> SEQ ID NO 21 <211>LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 21atgctaggtg accggatccc gcggataacc acca 34 <210> SEQ ID NO 22 <211>LENGTH: 58 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 22ccatgggaca tcatcaccat caccacgggg atccacaagc ttatgaagaa attagcaa 58 <210>SEQ ID NO 23 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: A synthetic primer<400> SEQUENCE: 23 ttctggatcc cgcggtatac caccacgaag actcaacac 39 <210>SEQ ID NO 24 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: A synthetic primer<400> SEQUENCE: 24 ttctggatcc cgcggcatac caccacgaag actcaacac 39 <210>SEQ ID NO 25 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: A synthetic primer<400> SEQUENCE: 25 ttctggatcc cgcggctcac caccacgaag actcaacac 39 <210>SEQ ID NO 26 <211> LENGTH: 118 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: A syntheticprimer <400> SEQUENCE: 26 tatgggccct taatacgact cactataggg gaattgtgagcggataacaa ttcccctcta 60 gaaataattt tgtttaactt taagaaggag atataccatgcagattttcg tgaaaacc 118 <210> SEQ ID NO 27 <211> LENGTH: 43 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 27 ttttggcgtc ggtgaccggatcccgcggtc gaccaccacg aag 43 <210> SEQ ID NO 28 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 28 ttttggcgtc ggtgaccggatcccgcggtg caccaccacg aag 43 <210> SEQ ID NO 29 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 29 ttttggcgtc ggtgaccggatcccgcgggt taccaccacg aag 43 <210> SEQ ID NO 30 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 30 ttttggcgtc ggtgaccggatcccgcggat caccaccacg aag 43 <210> SEQ ID NO 31 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 31 ttttggcgtc ggtgaccggatcccgcggga aaccaccacg aag 43 <210> SEQ ID NO 32 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 32 ttttggcgtc ggtgaccggatcccgcggat gaccaccacg aag 43 <210> SEQ ID NO 33 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 33 ttttggcgtc ggtgaccggatcccgcgggt gaccaccacg aag 43 <210> SEQ ID NO 34 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 34 ttttggcgtc ggtgaccggatcccgcggga gaccaccacg aag 43 <210> SEQ ID NO 35 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 35 ttttggcgtc ggtgaccggatcccgcggct taccaccacg aag 43 <210> SEQ ID NO 36 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 36 ttttggcgtc ggtgaccggatcccgcggtt gaccaccacg aag 43 <210> SEQ ID NO 37 <211> LENGTH: 43 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 37 ttttggcgtc ggtgaccggatcccgcggcc aaccaccacg aag 43 <210> SEQ ID NO 38 <211> LENGTH: 37 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 38 gtttttggcg tcggtgacctcaccaccacg aagactc 37 <210> SEQ ID NO 39 <211> LENGTH: 37 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 39 gagtcttcgt ggtggtgaggtcaccgacgc caaaaac 37 <210> SEQ ID NO 40 <211> LENGTH: 24 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 40 gttccaggaa ccagggcgtatctc 24 <210> SEQ ID NO 41 <211> LENGTH: 24 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Asynthetic primer <400> SEQUENCE: 41 cgcggaggag ttgtgtttgt ggac 24 <210>SEQ ID NO 42 <211> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: A synthetic primer<400> SEQUENCE: 42 ggcgaagctt gggtcaccga tgctaagaac attaagaagg g 41<210> SEQ ID NO 43 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: A syntheticprimer <400> SEQUENCE: 43 gctctagaat gaattcacgg cgatcttgcc gcc 33 <210>SEQ ID NO 44 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: A synthetic primer<400> SEQUENCE: 44 agctagcgag gctggatcgg tcccggt 27 <210> SEQ ID NO 45<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: A synthetic primer <400>SEQUENCE: 45 gattaatggc cctttcgtcc tcgagtt 27 <210> SEQ ID NO 46 <211>LENGTH: 174 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 46gcttgcaaga actggttcag tagcttaagc cactttgtga tccaccttaa cagccacggc 60ttccctcccg aggtggagga gcaggccgcc ggcaccctgc ccatgagctg cgcccaggag 120agcggcatgg atagacaccc tgctgcttgc gccagcgcca ggatcaacgt ctag 174 <210>SEQ ID NO 47 <211> LENGTH: 936 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: A syntheticoptimized Renilla luciferase DNA <400> SEQUENCE: 47 atggcttccaaggtgtacga ccccgagcaa cgcaaacgca tgatcactgg gcctcagtgg 60 tgggctcgctgcaagcaaat gaacgtgctg gactccttca tcaactacta tgattccgag 120 aagcacgccgagaacgccgt gatttttctg catggtaacg ctgcctccag ctacctgtgg 180 aggcacgtcgtgcctcacat cgagcccgtg gctagatgca tcatccctga tctgatcgga 240 atgggtaagtccggcaagag cgggaatggc tcatatcgcc tcctggatca ctacaagtac 300 ctcaccgcttggttcgagct gctgaacctt ccaaagaaaa tcatctttgt gggccacgac 360 tggggggcttgtctggcctt tcactactcc tacgagcacc aagacaagat caaggccatc 420 gtccatgctgagagtgtcgt ggacgtgatc gagtcctggg acgagtggcc tgacatcgag 480 gaggatatcgccctgatcaa gagcgaagag ggcgagaaaa tggtgcttga gaataacttc 540 ttcgtcgagaccatgctccc aagcaagatc atgcggaaac tggagcctga ggagttcgct 600 gcctacctggagccattcaa ggagaagggc gaggttagac ggcctaccct ctcctggcct 660 cgcgagatccctctcgttaa gggaggcaag cccgacgtcg tccagattgt ccgcaactac 720 aacgcctaccttcgggccag cgacgatctg cctaagatgt tcatcgagtc cgaccctggg 780 ttcttttccaacgctattgt cgagggagct aagaagttcc ctaacaccga gttcgtgaag 840 gtgaagggcctccacttcag ccaggaggac gctccagatg aaatgggtaa gtacatcaag 900 agcttcgtggagcgcgtgct gaagaacgag cagtaa 936 <210> SEQ ID NO 48 <211> LENGTH: 1653<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: A synthetic optimized firefly luciferase DNA <400>SEQUENCE: 48 atggccgatg ctaagaacat taagaagggc cctgctccct tctaccctctggaggatggc 60 accgctggcg agcagctgca caaggccatg aagaggtatg ccctggtgcctggcaccatt 120 gccttcaccg atgcccacat tgaggtggac atcacctatg ccgagtacttcgagatgtct 180 gtgcgcctgg ccgaggccat gaagaggtac ggcctgaaca ccaaccaccgcatcgtggtg 240 tgctctgaga actctctgca gttcttcatg ccagtgctgg gcgccctgttcatcggagtg 300 gccgtggccc ctgctaacga catttacaac gagcgcgagc tgctgaacagcatgggcatt 360 tctcagccta ccgtggtgtt cgtgtctaag aagggcctgc agaagatcctgaacgtgcag 420 aagaagctgc ctatcatcca gaagatcatc atcatggact ctaagaccgactaccagggc 480 ttccagagca tgtacacatt cgtgacatct catctgcctc ctggcttcaacgagtacgac 540 ttcgtgccag agtctttcga cagggacaaa accattgccc tgatcatgaacagctctggg 600 tctaccggcc tgcctaaggg cgtggccctg cctcatcgca ccgcctgtgtgcgcttctct 660 cacgcccgcg accctatttt cggcaaccag atcatccccg acaccgctattctgagcgtg 720 gtgccattcc accacggctt cggcatgttc accaccctgg gctacctgatttgcggcttt 780 cgggtggtgc tgatgtaccg cttcgaggag gagctgttcc tgcgcagcctgcaagactac 840 aaaattcagt ctgccctgct ggtgccaacc ctgttcagct tcttcgctaagagcaccctg 900 atcgacaagt acgacctgtc taacctgcac gagattgcct ctggcggcgccccactgtct 960 aaggaggtgg gcgaagccgt ggccaagcgc tttcatctgc caggcatccgccagggctac 1020 ggcctgaccg agacaaccag cgccattctg attaccccag agggcgacgacaagcctggc 1080 gccgtgggca aggtggtgcc attcttcgag gccaaggtgg tggacctggacaccggcaag 1140 accctgggag tgaaccagcg cggcgagctg tgtgtgcgcg gccctatgattatgtccggc 1200 tacgtgaata accctgaggc cacaaacgcc ctgatcgaca aggacggctggctgcactct 1260 ggcgacattg cctactggga cgaggacgag cacttcttca tcgtggaccgcctgaagtct 1320 ctgatcaagt acaagggcta ccaggtggcc ccagccgagc tggagtctatcctgctgcag 1380 caccctaaca ttttcgacgc cggagtggcc ggcctgcccg acgacgatgccggcgagctg 1440 cctgccgccg tcgtcgtgct ggaacacggc aagaccatga ccgagaaggagatcgtggac 1500 tatgtggcca gccaggtgac aaccgccaag aagctgcgcg gcggagtggtgttcgtggac 1560 gaggtgccca agggcctgac cggcaagctg gacgcccgca agatccgcgagatcctgatc 1620 aaggctaaga aaggcggcaa gatcgccgtg taa 1653 <210> SEQ IDNO 49 <211> LENGTH: 1653 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: A synthetic optimizedmutant firefly luciferase DNA <400> SEQUENCE: 49 atggccgatg ctaagaacattaagaagggc cctgctccct tctaccctct ggaggatggc 60 accgctggcg agcagctgcacaaggccatg aagaggtatg ccctggtgcc tggcaccatt 120 gccttcaccg atgcccacattgaggtggac atcacctatg ccgagtactt cgagatgtct 180 gtgcgcctgg ccgaggccatgaagaggtac ggcctgaaca ccaaccaccg catcgtggtg 240 tgctctgaga actctctgcagttcttcatg ccagtgctgg gcgccctgtt catcggagtg 300 gccgtggccc ctgctaacgacatttacaac gagcgcgagc tgctgaacag catgggcatt 360 tctcagccta ccgtggtgttcgtgtctaag aagggcctgc agaagatcct gaacgtgcag 420 aagaagctgc ctatcatccagaagatcatc atcatggact ctaagaccga ctaccagggc 480 ttccagagca tgtacacattcgtgacatct catctgcctc ctggcttcaa cgagtacgac 540 ttcgtgccag agtctttcgacagggacaaa accattgccc tgatcatgaa cagctctggg 600 tctaccggcc tgcctaagggcgtggccctg acccatcgca acgcctgtgt gcgcttctct 660 cacgcccgcg accctattttcggcaaccag atcatccccg acaccgctat tctgagcgtg 720 gtgccattcc accacggcttcggcatgttc accaccctgg gctacctgat ttgcggcttt 780 cgggtggtgc tgatgtaccgcttcgaggag gagctgttcc tgcgcagcct gcaagactac 840 aaaattcagt ctgccctgctggtgccaacc ctgttcagct tcttcgctaa gagcaccctg 900 atcgacaagt acgacctgtctaacctgcac gagattgcct ctggcggcgc cccactgtct 960 aaggaggtgg gcgaagccgtggccaagcgc tttcatctgc caggcatccg ccagggctac 1020 ggcctgaccg agacaaccagcgccattctg attaccccag agggcgacga caagcctggc 1080 gccgtgggca aggtggtgccattcttcgag gccaaggtgg tggacctgga caccggcaag 1140 accctgggag tgaaccagcgcggcgagctg tgtgtgcgcg gccctatgat tatgtccggc 1200 tacgtgaata accctgaggccacaaacgcc ctgatcgaca aggacggctg gctgcactct 1260 ggcgacattg cctactgggacgaggacgag cacttcttca tcgtggaccg cctgaagtct 1320 ctgatcaagt acaagggctaccaggtggcc ccagccgagc tggagtctat cctgctgcag 1380 caccctaaca ttttcgacgccggagtggcc ggcctgcccg acgacgatgc cggcgagctg 1440 cctgccgccg tcgtcgtgctggaacacggc aagaccatga ccgagaagga gatcgtggac 1500 tatgtggcca gccaggtgacaaccgccaag aagctgcgcg gcggagtggt gttcgtggac 1560 gaggtgccca agggcctgaccggcaagctg gacgcccgca agatccgcga gatcctgatc 1620 aaggctaaga aaggcggcaagatcgccgtg taa 1653 <210> SEQ ID NO 50 <211> LENGTH: 28 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 50 gattaatggc cctttcgtccttcgagtt 28 <210> SEQ ID NO 51 <211> LENGTH: 27 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Asynthetic primer <400> SEQUENCE: 51 agctagcgag gctggatcgg tcccggt 27<210> SEQ ID NO 52 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: A syntheticprimer <400> SEQUENCE: 52 ctagatttat ttatttattt cttcatatgc 30 <210> SEQID NO 53 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: A synthetic primer<400> SEQUENCE: 53 aattgcatat gaagaaataa ataaataaat 30 <210> SEQ ID NO54 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: A synthetic primer <400>SEQUENCE: 54 attaatctga tcaataaagg gtttaagg 28 <210> SEQ ID NO 55 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 55aaaaaggtag tggactgtcg 20 <210> SEQ ID NO 56 <211> LENGTH: 71 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 56 aattgggaat taaaacagcattgaaccaag aagcttggct ttcttatcaa ttctttgtga 60 cataataagt t 71 <210> SEQID NO 57 <211> LENGTH: 67 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: A synthetic primer<400> SEQUENCE: 57 aacttattat gtcacaaaga attgataaga aagccaagcttcttggttca atgctgtttt 60 aattccc 67 <210> SEQ ID NO 58 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 58 gatctgcggccgcatatatg 20 <210> SEQ ID NO 59 <211> LENGTH: 21 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Asynthetic primer <400> SEQUENCE: 59 gtgaccatat atgcggccgc a 21 <210> SEQID NO 60 <211> LENGTH: 57 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: A synthetic primer<400> SEQUENCE: 60 aatttgtctg cctgcaagaa ctggttcagc agcttgagccacttcgtgat ccacttg 57 <210> SEQ ID NO 61 <211> LENGTH: 57 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 61 aattcaagtg gatcacgaagtggctcaagc tgctgaacca gttcttgcag gcagaca 57 <210> SEQ ID NO 62 <211>LENGTH: 59 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: A synthetic primer <400> SEQUENCE: 62aattctgcct gcaagaactg gttcagcagc ttgagccact tcgtgatcca cttgtaagc 59<210> SEQ ID NO 63 <211> LENGTH: 59 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: A syntheticprimer <400> SEQUENCE: 63 ggccgcttac aagtggatca cgaagtggct caagctgctgaaccagttct tgcaggcag 59 <210> SEQ ID NO 64 <211> LENGTH: 62 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic primer <400> SEQUENCE: 64 gatcttatgt ctgcctgcaagaactggttc agcagcttga gccacttcgt gatccacttg 60 ca 62 <210> SEQ ID NO 65<211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: A synthetic primer <400>SEQUENCE: 65 agcttgcaag tggatcacga agtggctcaa gctgctgaac cagttcttgcaggcagacat 60 aa 62 <210> SEQ ID NO 66 <211> LENGTH: 1653 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic optimized firefly luciferase sequence <400>SEQUENCE: 66 atggccgatg ctaagaacat taagaagggc cctgctccct tctaccctctggaggatggc 60 accgctggcg agcagctgca caaggccatg aagaggtatg ccctggtgcctggcaccatt 120 gccttcaccg atgcccacat tgaggtggac atcacctatg ccgagtacttcgagatgtct 180 gtgcgcctgg ccgaggccat gaagaggtac ggcctgaaca ccaaccaccgcatcgtggtg 240 tgctctgaga actctctgca gttcttcatg ccagtgctgg gcgccctgttcatcggagtg 300 gccgtggccc ctgctaacga catttacaac gagcgcgagc tgctgaacagcatgggcatt 360 tctcagccta ccgtggtgtt cgtgtctaag aagggcctgc agaagatcctgaacgtgcag 420 aagaagctgc ctatcatcca gaagatcatc atcatggact ctaagaccgactaccagggc 480 ttccagagca tgtacacatt cgtgacatct catctgcctc ctggcttcaacgagtacgac 540 ttcgtgccag agtctttcga cagggacaaa accattgccc tgatcatgaacagctctggg 600 tctaccggcc tgcctaaggg cgtggccctg ccccatcgca ccgcctgtgtgcgcttctct 660 cacgcccgcg accctatttt cggcaaccag atcatccccg acaccgctattctgagcgtg 720 gtgccattcc accacggctt cggcatgttc accaccctgg gctacctgatttgcggcttt 780 cgggtggtgc tgatgtaccg cttcgaggag gagctgttcc tgcgcagcctgcaagactac 840 aaaattcagt ctgccctgct ggtgccaacc ctgttcagct tcttcgctaagagcaccctg 900 atcgacaagt acgacctgtc taacctgcac gagattgcct ctggcggcgccccactgtct 960 aaggaggtgg gcgaagccgt ggccaagcgc tttcatctgc caggcatccgccagggctac 1020 ggcctgaccg agacaaccag cgccattctg attaccccag agggcgacgacaagcctggc 1080 gccgtgggca aggtggtgcc attcttcgag gccaaggtgg tggacctggacaccggcaag 1140 accctgggag tgaaccagcg cggcgagctg tgtgtgcgcg gccctatgattatgtccggc 1200 tacgtgaata accctgaggc cacaaacgcc ctgatcgaca aggacggctggctgcactct 1260 ggcgacattg cctactggga cgaggacgag cacttcttca tcgtggaccgcctgaagtct 1320 ctgatcaagt acaagggcta ccaggtggcc ccagccgagc tggagtctatcctgctgcag 1380 caccctaaca ttttcgacgc cggagtggcc ggcctgcccg acgacgatgccggcgagctg 1440 cctgccgccg tcgtcgtgct ggaacacggc aagaccatga ccgagaaggagatcgtggac 1500 tatgtggcca gccaggtgac aaccgccaag aagctgcgcg gcggagtggtgttcgtggac 1560 gaggtgccca agggcctgac cggcaagctg gacgcccgca agatccgcgagatcctgatc 1620 aaggctaaga aaggcggcaa gatcgccgtg taa 1653 <210> SEQ IDNO 67 <400> SEQUENCE: 67 000 <210> SEQ ID NO 68 <211> LENGTH: 684 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic optimized GFP sequence <400> SEQUENCE: 68atgggcgtga tcaagcccga catgaagatc aagctgcgga tggagggcgc cgtgaacggc 60cacaaattcg tgatcgaggg cgacgggaaa ggcaagccct ttgagggtaa gcagactatg 120gacctgaccg tgatcgaggg cgcccccctg cccttcgctt atgacattct caccaccgtg 180ttcgactacg gtaaccgtgt cttcgccaag taccccaagg acatccctga ctacttcaag 240cagaccttcc ccgagggcta ctcgtgggag cgaagcatga catacgagga ccagggaatc 300tgtatcgcta caaacgacat caccatgatg aagggtgtgg acgactgctt cgtgtacaaa 360atccgcttcg acggggtcaa cttccctgct aatggcccgg tgatgcagcg caagacccta 420aagtgggagc ccagtaccga gaagatgtac gtgcgggacg gcgtactgaa gggcgatgtt 480aatatggcac tgctcttgga gggaggcggc cactaccgct gcgacttcaa gaccacctac 540aaagccaaga aggtggtgca gcttcccgac taccacttcg tggaccaccg catcgagatc 600gtgagccacg acaaggacta caacaaagtc aagctgtacg agcacgccga agcccacagc 660ggactacccc gccaggccgg ctaa 684 <210> SEQ ID NO 69 <211> LENGTH: 1776<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: A synthetic optimized firefly luciferase <400>SEQUENCE: 69 atggccgatg ctaagaacat taagaagggc cctgctccct tctaccctctggaggatggc 60 accgctggcg agcagctgca caaggccatg aagaggtatg ccctggtgcctggcaccatt 120 gccttcaccg atgcccacat tgaggtggac atcacctatg ccgagtacttcgagatgtct 180 gtgcgcctgg ccgaggccat gaagaggtac ggcctgaaca ccaaccaccgcatcgtggtg 240 tgctctgaga actctctgca gttcttcatg ccagtgctgg gcgccctgttcatcggagtg 300 gccgtggccc ctgctaacga catttacaac gagcgcgagc tgctgaacagcatgggcatt 360 tctcagccta ccgtggtgtt cgtgtctaag aagggcctgc agaagatcctgaacgtgcag 420 aagaagctgc ctatcatcca gaagatcatc atcatggact ctaagaccgactaccagggc 480 ttccagagca tgtacacatt cgtgacatct catctgcctc ctggcttcaacgagtacgac 540 ttcgtgccag agtctttcga cagggacaaa accattgccc tgatcatgaacagctctggg 600 tctaccggcc tgcctaaggg cgtggccctg acccatcgca acgcctgtgtgcgcttctct 660 cacgcccgcg accctatttt cggcaaccag atcatccccg acaccgctattctgagcgtg 720 gtgccattcc accacggctt cggcatgttc accaccctgg gctacctgatttgcggcttt 780 cgggtggtgc tgatgtaccg cttcgaggag gagctgttcc tgcgcagcctgcaagactac 840 aaaattcagt ctgccctgct ggtgccaacc ctgttcagct tcttcgctaagagcaccctg 900 atcgacaagt acgacctgtc taacctgcac gagattgcct ctggcggcgccccactgtct 960 aaggaggtgg gcgaagccgt ggccaagcgc tttcatctgc caggcatccgccagggctac 1020 ggcctgaccg agacaaccag cgccattctg attaccccag agggcgacgacaagcctggc 1080 gccgtgggca aggtggtgcc attcttcgag gccaaggtgg tggacctggacaccggcaag 1140 accctgggag tgaaccagcg cggcgagctg tgtgtgcgcg gccctatgattatgtccggc 1200 tacgtgaata accctgaggc cacaaacgcc ctgatcgaca aggacggctggctgcactct 1260 ggcgacattg cctactggga cgaggacgag cacttcttca tcgtggaccgcctgaagtct 1320 ctgatcaagt acaagggcta ccaggtggcc ccagccgagc tggagtctatcctgctgcag 1380 caccctaaca ttttcgacgc cggagtggcc ggcctgcccg acgacgatgccggcgagctg 1440 cctgccgccg tcgtcgtgct ggaacacggc aagaccatga ccgagaaggagatcgtggac 1500 tatgtggcca gccaggtgac aaccgccaag aagctgcgcg gcggagtggtgttcgtggac 1560 gaggtgccca agggcctgac cggcaagctg gacgcccgca agatccgcgagatcctgatc 1620 aaggctaaga aaggcggcaa gatcgccgtg aattctcacg gcttccctcccgaggtggag 1680 gagcaggccg ccggcaccct gcccatgagc tgcgcccagg agagcggcatggatagacac 1740 cctgctgctt gcgccagcgc caggatcaac gtctaa 1776 <210> SEQID NO 70 <211> LENGTH: 1829 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: A synthetic optimizedfirefly luciferase <400> SEQUENCE: 70 atggccgatg ctaagaacat taagaagggccctgctccct tctaccctct ggaggatggc 60 accgctggcg agcagctgca caaggccatgaagaggtatg ccctggtgcc tggcaccatt 120 gccttcaccg atgcccacat tgaggtggacatcacctatg ccgagtactt cgagatgtct 180 gtgcgcctgg ccgaggccat gaagaggtacggcctgaaca ccaaccaccg catcgtggtg 240 tgctctgaga actctctgca gttcttcatgccagtgctgg gcgccctgtt catcggagtg 300 gccgtggccc ctgctaacga catttacaacgagcgcgagc tgctgaacag catgggcatt 360 tctcagccta ccgtggtgtt cgtgtctaagaagggcctgc agaagatcct gaacgtgcag 420 aagaagctgc ctatcatcca gaagatcatcatcatggact ctaagaccga ctaccagggc 480 ttccagagca tgtacacatt cgtgacatctcatctgcctc ctggcttcaa cgagtacgac 540 ttcgtgccag agtctttcga cagggacaaaaccattgccc tgatcatgaa cagctctggg 600 tctaccggcc tgcctaaggg cgtggccctgacccatcgca acgcctgtgt gcgcttctct 660 cacgcccgcg accctatttt cggcaaccagatcatccccg acaccgctat tctgagcgtg 720 gtgccattcc accacggctt cggcatgttcaccaccctgg gctacctgat ttgcggcttt 780 cgggtggtgc tgatgtaccg cttcgaggaggagctgttcc tgcgcagcct gcaagactac 840 aaaattcagt ctgccctgct ggtgccaaccctgttcagct tcttcgctaa gagcaccctg 900 atcgacaagt acgacctgtc taacctgcacgagattgcct ctggcggcgc cccactgtct 960 aaggaggtgg gcgaagccgt ggccaagcgctttcatctgc caggcatccg ccagggctac 1020 ggcctgaccg agacaaccag cgccattctgattaccccag agggcgacga caagcctggc 1080 gccgtgggca aggtggtgcc attcttcgaggccaaggtgg tggacctgga caccggcaag 1140 accctgggag tgaaccagcg cggcgagctgtgtgtgcgcg gccctatgat tatgtccggc 1200 tacgtgaata accctgaggc cacaaacgccctgatcgaca aggacggctg gctgcactct 1260 ggcgacattg cctactggga cgaggacgagcacttcttca tcgtggaccg cctgaagtct 1320 ctgatcaagt acaagggcta ccaggtggccccagccgagc tggagtctat cctgctgcag 1380 caccctaaca ttttcgacgc cggagtggccggcctgcccg acgacgatgc cggcgagctg 1440 cctgccgccg tcgtcgtgct ggaacacggcaagaccatga ccgagaagga gatcgtggac 1500 tatgtggcca gccaggtgac aaccgccaagaagctgcgcg gcggagtggt gttcgtggac 1560 gaggtgccca agggcctgac cggcaagctggacgcccgca agatccgcga gatcctgatc 1620 aaggctaaga aaggcggcaa gatcgccgtgaattctgctt gcaagaactg gttcagtagc 1680 ttaagccact ttgtgatcca ccttaacagccacggcttcc ctcccgaggt ggaggagcag 1740 gccgccggca ccctgcccat gagctgcgcccaggagagcg gcatggatag acaccctgct 1800 gcttgcgcca gcgccaggat caacgtcta1829 <210> SEQ ID NO 71 <211> LENGTH: 1776 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Asynthetic optimized firefly luciferase <400> SEQUENCE: 71 atggccgatgctaagaacat taagaagggc cctgctccct tctaccctct ggaggatggc 60 accgctggcgagcagctgca caaggccatg aagaggtatg ccctggtgcc tggcaccatt 120 gccttcaccgatgcccacat tgaggtggac atcacctatg ccgagtactt cgagatgtct 180 gtgcgcctggccgaggccat gaagaggtac ggcctgaaca ccaaccaccg catcgtggtg 240 tgctctgagaactctctgca gttcttcatg ccagtgctgg gcgccctgtt catcggagtg 300 gccgtggcccctgctaacga catttacaac gagcgcgagc tgctgaacag catgggcatt 360 tctcagcctaccgtggtgtt cgtgtctaag aagggcctgc agaagatcct gaacgtgcag 420 aagaagctgcctatcatcca gaagatcatc atcatggact ctaagaccga ctaccagggc 480 ttccagagcatgtacacatt cgtgacatct catctgcctc ctggcttcaa cgagtacgac 540 ttcgtgccagagtctttcga cagggacaaa accattgccc tgatcatgaa cagctctggg 600 tctaccggcctgcctaaggg cgtggccctg cctcatcgca ccgcctgtgt gcgcttctct 660 cacgcccgcgaccctatttt cggcaaccag atcatccccg acaccgctat tctgagcgtg 720 gtgccattccaccacggctt cggcatgttc accaccctgg gctacctgat ttgcggcttt 780 cgggtggtgctgatgtaccg cttcgaggag gagctgttcc tgcgcagcct gcaagactac 840 aaaattcagtctgccctgct ggtgccaacc ctgttcagct tcttcgctaa gagcaccctg 900 atcgacaagtacgacctgtc taacctgcac gagattgcct ctggcggcgc cccactgtct 960 aaggaggtgggcgaagccgt ggccaagcgc tttcatctgc caggcatccg ccagggctac 1020 ggcctgaccgagacaaccag cgccattctg attaccccag agggcgacga caagcctggc 1080 gccgtgggcaaggtggtgcc attcttcgag gccaaggtgg tggacctgga caccggcaag 1140 accctgggagtgaaccagcg cggcgagctg tgtgtgcgcg gccctatgat tatgtccggc 1200 tacgtgaataaccctgaggc cacaaacgcc ctgatcgaca aggacggctg gctgcactct 1260 ggcgacattgcctactggga cgaggacgag cacttcttca tcgtggaccg cctgaagtct 1320 ctgatcaagtacaagggcta ccaggtggcc ccagccgagc tggagtctat cctgctgcag 1380 caccctaacattttcgacgc cggagtggcc ggcctgcccg acgacgatgc cggcgagctg 1440 cctgccgccgtcgtcgtgct ggaacacggc aagaccatga ccgagaagga gatcgtggac 1500 tatgtggccagccaggtgac aaccgccaag aagctgcgcg gcggagtggt gttcgtggac 1560 gaggtgcccaagggcctgac cggcaagctg gacgcccgca agatccgcga gatcctgatc 1620 aaggctaagaaaggcggcaa gatcgccgtg aattctcacg gcttccctcc cgaggtggag 1680 gagcaggccgccggcaccct gcccatgagc tgcgcccagg agagcggcat ggatagacac 1740 cctgctgcttgcgccagcgc caggatcaac gtctaa 1776 <210> SEQ ID NO 72 <211> LENGTH: 1830<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: A synthetic optimized firefly luciferase <400>SEQUENCE: 72 atggccgatg ctaagaacat taagaagggc cctgctccct tctaccctctggaggatggc 60 accgctggcg agcagctgca caaggccatg aagaggtatg ccctggtgcctggcaccatt 120 gccttcaccg atgcccacat tgaggtggac atcacctatg ccgagtacttcgagatgtct 180 gtgcgcctgg ccgaggccat gaagaggtac ggcctgaaca ccaaccaccgcatcgtggtg 240 tgctctgaga actctctgca gttcttcatg ccagtgctgg gcgccctgttcatcggagtg 300 gccgtggccc ctgctaacga catttacaac gagcgcgagc tgctgaacagcatgggcatt 360 tctcagccta ccgtggtgtt cgtgtctaag aagggcctgc agaagatcctgaacgtgcag 420 aagaagctgc ctatcatcca gaagatcatc atcatggact ctaagaccgactaccagggc 480 ttccagagca tgtacacatt cgtgacatct catctgcctc ctggcttcaacgagtacgac 540 ttcgtgccag agtctttcga cagggacaaa accattgccc tgatcatgaacagctctggg 600 tctaccggcc tgcctaaggg cgtggccctg cctcatcgca ccgcctgtgtgcgcttctct 660 cacgcccgcg accctatttt cggcaaccag atcatccccg acaccgctattctgagcgtg 720 gtgccattcc accacggctt cggcatgttc accaccctgg gctacctgatttgcggcttt 780 cgggtggtgc tgatgtaccg cttcgaggag gagctgttcc tgcgcagcctgcaagactac 840 aaaattcagt ctgccctgct ggtgccaacc ctgttcagct tcttcgctaagagcaccctg 900 atcgacaagt acgacctgtc taacctgcac gagattgcct ctggcggcgccccactgtct 960 aaggaggtgg gcgaagccgt ggccaagcgc tttcatctgc caggcatccgccagggctac 1020 ggcctgaccg agacaaccag cgccattctg attaccccag agggcgacgacaagcctggc 1080 gccgtgggca aggtggtgcc attcttcgag gccaaggtgg tggacctggacaccggcaag 1140 accctgggag tgaaccagcg cggcgagctg tgtgtgcgcg gccctatgattatgtccggc 1200 tacgtgaata accctgaggc cacaaacgcc ctgatcgaca aggacggctggctgcactct 1260 ggcgacattg cctactggga cgaggacgag cacttcttca tcgtggaccgcctgaagtct 1320 ctgatcaagt acaagggcta ccaggtggcc ccagccgagc tggagtctatcctgctgcag 1380 caccctaaca ttttcgacgc cggagtggcc ggcctgcccg acgacgatgccggcgagctg 1440 cctgccgccg tcgtcgtgct ggaacacggc aagaccatga ccgagaaggagatcgtggac 1500 tatgtggcca gccaggtgac aaccgccaag aagctgcgcg gcggagtggtgttcgtggac 1560 gaggtgccca agggcctgac cggcaagctg gacgcccgca agatccgcgagatcctgatc 1620 aaggctaaga aaggcggcaa gatcgccgtg aattctgctt gcaagaactggttcagtagc 1680 ttaagccact ttgtgatcca ccttaacagc cacggcttcc ctcccgaggtggaggagcag 1740 gccgccggca ccctgcccat gagctgcgcc caggagagcg gcatggatagacaccctgct 1800 gcttgcgcca gcgccaggat caacgtctag 1830 <210> SEQ ID NO 73<211> LENGTH: 1059 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: A synthetic optimized Renillaluciferase <400> SEQUENCE: 73 atggcttcca aggtgtacga ccccgagcaacgcaaacgca tgatcactgg gcctcagtgg 60 tgggctcgct gcaagcaaat gaacgtgctggactccttca tcaactacta tgattccgag 120 aagcacgccg agaacgccgt gatttttctgcatggtaacg ctgcctccag ctacctgtgg 180 aggcacgtcg tgcctcacat cgagcccgtggctagatgca tcatccctga tctgatcgga 240 atgggtaagt ccggcaagag cgggaatggctcatatcgcc tcctggatca ctacaagtac 300 ctcaccgctt ggttcgagct gctgaaccttccaaagaaaa tcatctttgt gggccacgac 360 tggggggctt gtctggcctt tcactactcctacgagcacc aagacaagat caaggccatc 420 gtccatgctg agagtgtcgt ggacgtgatcgagtcctggg acgagtggcc tgacatcgag 480 gaggatatcg ccctgatcaa gagcgaagagggcgagaaaa tggtgcttga gaataacttc 540 ttcgtcgaga ccatgctccc aagcaagatcatgcggaaac tggagcctga ggagttcgct 600 gcctacctgg agccattcaa ggagaagggcgaggttagac ggcctaccct ctcctggcct 660 cgcgagatcc ctctcgttaa gggaggcaagcccgacgtcg tccagattgt ccgcaactac 720 aacgcctacc ttcgggccag cgacgatctgcctaagatgt tcatcgagtc cgaccctggg 780 ttcttttcca acgctattgt cgagggagctaagaagttcc ctaacaccga gttcgtgaag 840 gtgaagggcc tccacttcag ccaggaggacgctccagatg aaatgggtaa gtacatcaag 900 agcttcgtgg agcgcgtgct gaagaacgagcagaattctc acggcttccc tcccgaggtg 960 gaggagcagg ccgccggcac cctgcccatgagctgcgccc aggagagcgg catggataga 1020 caccctgctg cttgcgccag cgccaggatcaacgtctaa 1059 <210> SEQ ID NO 74 <211> LENGTH: 1113 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: A synthetic optimized Renilla luciferase <400> SEQUENCE: 74atggcttcca aggtgtacga ccccgagcaa cgcaaacgca tgatcactgg gcctcagtgg 60tgggctcgct gcaagcaaat gaacgtgctg gactccttca tcaactacta tgattccgag 120aagcacgccg agaacgccgt gatttttctg catggtaacg ctgcctccag ctacctgtgg 180aggcacgtcg tgcctcacat cgagcccgtg gctagatgca tcatccctga tctgatcgga 240atgggtaagt ccggcaagag cgggaatggc tcatatcgcc tcctggatca ctacaagtac 300ctcaccgctt ggttcgagct gctgaacctt ccaaagaaaa tcatctttgt gggccacgac 360tggggggctt gtctggcctt tcactactcc tacgagcacc aagacaagat caaggccatc 420gtccatgctg agagtgtcgt ggacgtgatc gagtcctggg acgagtggcc tgacatcgag 480gaggatatcg ccctgatcaa gagcgaagag ggcgagaaaa tggtgcttga gaataacttc 540ttcgtcgaga ccatgctccc aagcaagatc atgcggaaac tggagcctga ggagttcgct 600gcctacctgg agccattcaa ggagaagggc gaggttagac ggcctaccct ctcctggcct 660cgcgagatcc ctctcgttaa gggaggcaag cccgacgtcg tccagattgt ccgcaactac 720aacgcctacc ttcgggccag cgacgatctg cctaagatgt tcatcgagtc cgaccctggg 780ttcttttcca acgctattgt cgagggagct aagaagttcc ctaacaccga gttcgtgaag 840gtgaagggcc tccacttcag ccaggaggac gctccagatg aaatgggtaa gtacatcaag 900agcttcgtgg agcgcgtgct gaagaacgag cagaattctg cttgcaagaa ctggttcagt 960agcttaagcc actttgtgat ccaccttaac agccacggct tccctcccga ggtggaggag 1020caggccgccg gcaccctgcc catgagctgc gcccaggaga gcggcatgga tagacaccct 1080gctgcttgcg ccagcgccag gatcaacgtc tag 1113 <210> SEQ ID NO 75 <211>LENGTH: 1140 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: A synthetic optimized Renillaluciferase <400> SEQUENCE: 75 atggcttcca aggtgtacga ccccgagcaacgcaaacgca tgatcactgg gcctcagtgg 60 tgggctcgct gcaagcaaat gaacgtgctggactccttca tcaactacta tgattccgag 120 aagcacgccg agaacgccgt gatttttctgcatggtaacg ctgcctccag ctacctgtgg 180 aggcacgtcg tgcctcacat cgagcccgtggctagatgca tcatccctga tctgatcgga 240 atgggtaagt ccggcaagag cgggaatggctcatatcgcc tcctggatca ctacaagtac 300 ctcaccgctt ggttcgagct gctgaaccttccaaagaaaa tcatctttgt gggccacgac 360 tggggggctt gtctggcctt tcactactcctacgagcacc aagacaagat caaggccatc 420 gtccatgctg agagtgtcgt ggacgtgatcgagtcctggg acgagtggcc tgacatcgag 480 gaggatatcg ccctgatcaa gagcgaagagggcgagaaaa tggtgcttga gaataacttc 540 ttcgtcgaga ccatgctccc aagcaagatcatgcggaaac tggagcctga ggagttcgct 600 gcctacctgg agccattcaa ggagaagggcgaggttagac ggcctaccct ctcctggcct 660 cgcgagatcc ctctcgttaa gggaggcaagcccgacgtcg tccagattgt ccgcaactac 720 aacgcctacc ttcgggccag cgacgatctgcctaagatgt tcatcgagtc cgaccctggg 780 ttcttttcca acgctattgt cgagggagctaagaagttcc ctaacaccga gttcgtgaag 840 gtgaagggcc tccacttcag ccaggaggacgctccagatg aaatgggtaa gtacatcaag 900 agcttcgtgg agcgcgtgct gaagaacgagcagaattctg cttgcaagaa ctggttcagt 960 agcttaagcc actttgtgat ccaccttaacagccacggct tccctcccga ggtggaggag 1020 caggccgccg gcaccctgcc catgagctgcgcccaggaga gcggcatgga tagacaccct 1080 gctgcttgcg ccagcgccag gatcaacgtctagggcgcgg actttattta tttatttctt 1140 <210> SEQ ID NO 76 <211> LENGTH:1857 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: A synthetic optimized firefly luciferase <400>SEQUENCE: 76 atggccgatg ctaagaacat taagaagggc cctgctccct tctaccctctggaggatggc 60 accgctggcg agcagctgca caaggccatg aagaggtatg ccctggtgcctggcaccatt 120 gccttcaccg atgcccacat tgaggtggac atcacctatg ccgagtacttcgagatgtct 180 gtgcgcctgg ccgaggccat gaagaggtac ggcctgaaca ccaaccaccgcatcgtggtg 240 tgctctgaga actctctgca gttcttcatg ccagtgctgg gcgccctgttcatcggagtg 300 gccgtggccc ctgctaacga catttacaac gagcgcgagc tgctgaacagcatgggcatt 360 tctcagccta ccgtggtgtt cgtgtctaag aagggcctgc agaagatcctgaacgtgcag 420 aagaagctgc ctatcatcca gaagatcatc atcatggact ctaagaccgactaccagggc 480 ttccagagca tgtacacatt cgtgacatct catctgcctc ctggcttcaacgagtacgac 540 ttcgtgccag agtctttcga cagggacaaa accattgccc tgatcatgaacagctctggg 600 tctaccggcc tgcctaaggg cgtggccctg cctcatcgca ccgcctgtgtgcgcttctct 660 cacgcccgcg accctatttt cggcaaccag atcatccccg acaccgctattctgagcgtg 720 gtgccattcc accacggctt cggcatgttc accaccctgg gctacctgatttgcggcttt 780 cgggtggtgc tgatgtaccg cttcgaggag gagctgttcc tgcgcagcctgcaagactac 840 aaaattcagt ctgccctgct ggtgccaacc ctgttcagct tcttcgctaagagcaccctg 900 atcgacaagt acgacctgtc taacctgcac gagattgcct ctggcggcgccccactgtct 960 aaggaggtgg gcgaagccgt ggccaagcgc tttcatctgc caggcatccgccagggctac 1020 ggcctgaccg agacaaccag cgccattctg attaccccag agggcgacgacaagcctggc 1080 gccgtgggca aggtggtgcc attcttcgag gccaaggtgg tggacctggacaccggcaag 1140 accctgggag tgaaccagcg cggcgagctg tgtgtgcgcg gccctatgattatgtccggc 1200 tacgtgaata accctgaggc cacaaacgcc ctgatcgaca aggacggctggctgcactct 1260 ggcgacattg cctactggga cgaggacgag cacttcttca tcgtggaccgcctgaagtct 1320 ctgatcaagt acaagggcta ccaggtggcc ccagccgagc tggagtctatcctgctgcag 1380 caccctaaca ttttcgacgc cggagtggcc ggcctgcccg acgacgatgccggcgagctg 1440 cctgccgccg tcgtcgtgct ggaacacggc aagaccatga ccgagaaggagatcgtggac 1500 tatgtggcca gccaggtgac aaccgccaag aagctgcgcg gcggagtggtgttcgtggac 1560 gaggtgccca agggcctgac cggcaagctg gacgcccgca agatccgcgagatcctgatc 1620 aaggctaaga aaggcggcaa gatcgccgtg aattctgctt gcaagaactggttcagtagc 1680 ttaagccact ttgtgatcca ccttaacagc cacggcttcc ctcccgaggtggaggagcag 1740 gccgccggca ccctgcccat gagctgcgcc caggagagcg gcatggatagacaccctgct 1800 gcttgcgcca gcgccaggat caacgtctag ggcgcggact ttatttatttatttctt 1857 <210> SEQ ID NO 77 <211> LENGTH: 1752 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Asynthetic optimized click beetle sequence <400> SEQUENCE: 77 atggtaaagcgtgagaaaaa tgtcatctat ggccctgagc ctctccatcc tttggaggat 60 ttgactgccggcgaaatgct gtttcgtgct ctccgcaagc actctcattt gcctcaagcc 120 ttggtcgatgtggtcggcga tgaatctttg agctacaagg agttttttga ggcaaccgtc 180 ttgctggctcagtccctcca caattgtggc tacaagatga acgacgtcgt tagtatctgt 240 gctgaaaacaatacccgttt cttcattcca gtcatcgccg catggtatat cggtatgatc 300 gtggctccagtcaacgagag ctacattccc gacgaactgt gtaaagtcat gggtatctct 360 aagccacagattgtcttcac cactaagaat attctgaaca aagtcctgga agtccaaagc 420 cgcaccaactttattaagcg tatcatcatc ttggacactg tggagaatat tcacggttgc 480 gaatctttgcctaatttcat ctctcgctat tcagacggca acatcgcaaa ctttaaacca 540 ctccacttcgaccctgtgga acaagttgca gccattctgt gtagcagcgg tactactgga 600 ctcccaaagggagtcatgca gacccatcaa aacatttgcg tgcgtctgat ccatgctctc 660 gatccacgctacggcactca gctgattcct ggtgtcaccg tcttggtcta cttgcctttc 720 ttccatgctttcggctttca tattactttg ggttacttta tggtcggtct ccgcgtgatt 780 atgttccgccgttttgatca ggaggctttc ttgaaagcca tccaagatta tgaagtccgc 840 agtgtcatcaacgtgcctag cgtgatcctg tttttgtcta agagcccact cgtggacaag 900 tacgacttgtcttcactgcg tgaattgtgt tgcggtgccg ctccactggc taaggaggtc 960 gctgaagtggccgccaaacg cttgaatctt ccagggattc gttgtggctt cggcctcacc 1020 gaatctaccagtgcgattat ccagactctc ggggatgagt ttaagagcgg ctctttgggc 1080 cgtgtcactccactcatggc tgctaagatc gctgatcgcg aaactggtaa ggctttgggc 1140 ccgaaccaagtgggcgagct gtgtatcaaa ggccctatgg tgagcaaggg ttatgtcaat 1200 aacgttgaagctaccaagga ggccatcgac gacgacggct ggttgcattc tggtgatttt 1260 ggatattacgacgaagatga gcatttttac gtcgtggatc gttacaagga gctgatcaaa 1320 tacaagggtagccaggttgc tccagctgag ttggaggaga ttctgttgaa aaatccatgc 1380 attcgcgatgtcgctgtggt cggcattcct gatctggagg ccggcgaact gccttctgct 1440 ttcgttgtcaagcagcctgg tacagaaatt accgccaaag aagtgtatga ttacctggct 1500 gaacgtgtgagccatactaa gtacttgcgt ggcggcgtgc gttttgttga ctccatccct 1560 cgtaacgtaacaggcaaaat tacccgcaag gagctgttga aacaattgtt ggtgaaggcc 1620 ggcgggaattctcacggctt ccctcccgag gtggaggagc aggccgccgg caccctgccc 1680 atgagctgcgcccaggagag cggcatggat agacaccctg ctgcttgcgc cagcgccagg 1740 atcaacgtctaa 1752 <210> SEQ ID NO 78 <211> LENGTH: 1833 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Asynthetic optimized click beetle sequence <400> SEQUENCE: 78 atggtaaagcgtgagaaaaa tgtcatctat ggccctgagc ctctccatcc tttggaggat 60 ttgactgccggcgaaatgct gtttcgtgct ctccgcaagc actctcattt gcctcaagcc 120 ttggtcgatgtggtcggcga tgaatctttg agctacaagg agttttttga ggcaaccgtc 180 ttgctggctcagtccctcca caattgtggc tacaagatga acgacgtcgt tagtatctgt 240 gctgaaaacaatacccgttt cttcattcca gtcatcgccg catggtatat cggtatgatc 300 gtggctccagtcaacgagag ctacattccc gacgaactgt gtaaagtcat gggtatctct 360 aagccacagattgtcttcac cactaagaat attctgaaca aagtcctgga agtccaaagc 420 cgcaccaactttattaagcg tatcatcatc ttggacactg tggagaatat tcacggttgc 480 gaatctttgcctaatttcat ctctcgctat tcagacggca acatcgcaaa ctttaaacca 540 ctccacttcgaccctgtgga acaagttgca gccattctgt gtagcagcgg tactactgga 600 ctcccaaagggagtcatgca gacccatcaa aacatttgcg tgcgtctgat ccatgctctc 660 gatccacgctacggcactca gctgattcct ggtgtcaccg tcttggtcta cttgcctttc 720 ttccatgctttcggctttca tattactttg ggttacttta tggtcggtct ccgcgtgatt 780 atgttccgccgttttgatca ggaggctttc ttgaaagcca tccaagatta tgaagtccgc 840 agtgtcatcaacgtgcctag cgtgatcctg tttttgtcta agagcccact cgtggacaag 900 tacgacttgtcttcactgcg tgaattgtgt tgcggtgccg ctccactggc taaggaggtc 960 gctgaagtggccgccaaacg cttgaatctt ccagggattc gttgtggctt cggcctcacc 1020 gaatctaccagtgcgattat ccagactctc ggggatgagt ttaagagcgg ctctttgggc 1080 cgtgtcactccactcatggc tgctaagatc gctgatcgcg aaactggtaa ggctttgggc 1140 ccgaaccaagtgggcgagct gtgtatcaaa ggccctatgg tgagcaaggg ttatgtcaat 1200 aacgttgaagctaccaagga ggccatcgac gacgacggct ggttgcattc tggtgatttt 1260 ggatattacgacgaagatga gcatttttac gtcgtggatc gttacaagga gctgatcaaa 1320 tacaagggtagccaggttgc tccagctgag ttggaggaga ttctgttgaa aaatccatgc 1380 attcgcgatgtcgctgtggt cggcattcct gatctggagg ccggcgaact gccttctgct 1440 ttcgttgtcaagcagcctgg tacagaaatt accgccaaag aagtgtatga ttacctggct 1500 gaacgtgtgagccatactaa gtacttgcgt ggcggcgtgc gttttgttga ctccatccct 1560 cgtaacgtaacaggcaaaat tacccgcaag gagctgttga aacaattgtt ggtgaaggcc 1620 ggcgggaattctgcttgcaa gaactggttc agtagcttaa gccactttgt gatccacctt 1680 aacagccacggcttccctcc cgaggtggag gagcaggccg ccggcaccct gcccatgagc 1740 tgcgcccaggagagcggcat ggatagacac cctgctgctt gcgccagcgc caggatcaac 1800 gtctagggcgcggactttat ttatttattt ctt 1833 <210> SEQ ID NO 79 <211> LENGTH: 1752<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: A synthetic optimized click beetle sequence <400>SEQUENCE: 79 atggtgaagc gtgagaaaaa tgtcatctat ggccctgagc ctctccatcctttggaggat 60 ttgactgccg gcgaaatgct gtttcgtgct ctccgcaagc actctcatttgcctcaagcc 120 ttggtcgatg tggtcggcga tgaatctttg agctacaagg agttttttgaggcaaccgtc 180 ttgctggctc agtccctcca caattgtggc tacaagatga acgacgtcgttagtatctgt 240 gctgaaaaca atacccgttt cttcattcca gtcatcgccg catggtatatcggtatgatc 300 gtggctccag tcaacgagag ctacattccc gacgaactgt gtaaagtcatgggtatctct 360 aagccacaga ttgtcttcac cactaagaat attctgaaca aagtcctggaagtccaaagc 420 cgcaccaact ttattaagcg tatcatcatc ttggacactg tggagaatattcacggttgc 480 gaatctttgc ctaatttcat ctctcgctat tcagacggca acatcgcaaactttaaacca 540 ctccacttcg accctgtgga acaagttgca gccattctgt gtagcagcggtactactgga 600 ctcccaaagg gagtcatgca gacccatcaa aacatttgcg tgcgtctgatccatgctctc 660 gatccacgcg tgggcactca gctgattcct ggtgtcaccg tcttggtctacttgcctttc 720 ttccatgctt tcggctttag cattactttg ggttacttta tggtcggtctccgcgtgatt 780 atgttccgcc gttttgatca ggaggctttc ttgaaagcca tccaagattatgaagtccgc 840 agtgtcatca acgtgcctag cgtgatcctg tttttgtcta agagcccactcgtggacaag 900 tacgacttgt cttcactgcg tgaattgtgt tgcggtgccg ctccactggctaaggaggtc 960 gctgaagtgg ccgccaaacg cttgaatctt ccagggattc gttgtggcttcggcctcacc 1020 gaatctacca gcgctaacat tcactctctc ggggatgagt ttaagagcggctctttgggc 1080 cgtgtcactc cactcatggc tgctaagatc gctgatcgcg aaactggtaaggctttgggc 1140 ccgaaccaag tgggcgagct gtgtatcaaa ggccctatgg tgagcaagggttatgtcaat 1200 aacgttgaag ctaccaagga ggccatcgac gacgacggct ggttgcattctggtgatttt 1260 ggatattacg acgaagatga gcatttttac gtcgtggatc gttacaaggagctgatcaaa 1320 tacaagggta gccaggttgc tccagctgag ttggaggaga ttctgttgaaaaatccatgc 1380 attcgcgatg tcgctgtggt cggcattcct gatctggagg ccggcgaactgccttctgct 1440 ttcgttgtca agcagcctgg taaagaaatt accgccaaag aagtgtatgattacctggct 1500 gaacgtgtga gccatactaa gtacttgcgt ggcggcgtgc gttttgttgactccatccct 1560 cgtaacgtaa caggcaaaat tacccgcaag gagctgttga aacaattgttggagaaggcc 1620 ggcgggaatt ctcacggctt ccctcccgag gtggaggagc aggccgccggcaccctgccc 1680 atgagctgcg cccaggagag cggcatggat agacaccctg ctgcttgcgccagcgccagg 1740 atcaacgtct aa 1752 <210> SEQ ID NO 80 <211> LENGTH: 1833<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: A synthetic optimized click beetle sequence <400>SEQUENCE: 80 atggtgaagc gtgagaaaaa tgtcatctat ggccctgagc ctctccatcctttggaggat 60 ttgactgccg gcgaaatgct gtttcgtgct ctccgcaagc actctcatttgcctcaagcc 120 ttggtcgatg tggtcggcga tgaatctttg agctacaagg agttttttgaggcaaccgtc 180 ttgctggctc agtccctcca caattgtggc tacaagatga acgacgtcgttagtatctgt 240 gctgaaaaca atacccgttt cttcattcca gtcatcgccg catggtatatcggtatgatc 300 gtggctccag tcaacgagag ctacattccc gacgaactgt gtaaagtcatgggtatctct 360 aagccacaga ttgtcttcac cactaagaat attctgaaca aagtcctggaagtccaaagc 420 cgcaccaact ttattaagcg tatcatcatc ttggacactg tggagaatattcacggttgc 480 gaatctttgc ctaatttcat ctctcgctat tcagacggca acatcgcaaactttaaacca 540 ctccacttcg accctgtgga acaagttgca gccattctgt gtagcagcggtactactgga 600 ctcccaaagg gagtcatgca gacccatcaa aacatttgcg tgcgtctgatccatgctctc 660 gatccacgcg tgggcactca gctgattcct ggtgtcaccg tcttggtctacttgcctttc 720 ttccatgctt tcggctttag cattactttg ggttacttta tggtcggtctccgcgtgatt 780 atgttccgcc gttttgatca ggaggctttc ttgaaagcca tccaagattatgaagtccgc 840 agtgtcatca acgtgcctag cgtgatcctg tttttgtcta agagcccactcgtggacaag 900 tacgacttgt cttcactgcg tgaattgtgt tgcggtgccg ctccactggctaaggaggtc 960 gctgaagtgg ccgccaaacg cttgaatctt ccagggattc gttgtggcttcggcctcacc 1020 gaatctacca gcgctaacat tcactctctc ggggatgagt ttaagagcggctctttgggc 1080 cgtgtcactc cactcatggc tgctaagatc gctgatcgcg aaactggtaaggctttgggc 1140 ccgaaccaag tgggcgagct gtgtatcaaa ggccctatgg tgagcaagggttatgtcaat 1200 aacgttgaag ctaccaagga ggccatcgac gacgacggct ggttgcattctggtgatttt 1260 ggatattacg acgaagatga gcatttttac gtcgtggatc gttacaaggagctgatcaaa 1320 tacaagggta gccaggttgc tccagctgag ttggaggaga ttctgttgaaaaatccatgc 1380 attcgcgatg tcgctgtggt cggcattcct gatctggagg ccggcgaactgccttctgct 1440 ttcgttgtca agcagcctgg taaagaaatt accgccaaag aagtgtatgattacctggct 1500 gaacgtgtga gccatactaa gtacttgcgt ggcggcgtgc gttttgttgactccatccct 1560 cgtaacgtaa caggcaaaat tacccgcaag gagctgttga aacaattgttggagaaggcc 1620 ggcgggaatt ctgcttgcaa gaactggttc agtagcttaa gccactttgtgatccacctt 1680 aacagccacg gcttccctcc cgaggtggag gagcaggccg ccggcaccctgcccatgagc 1740 tgcgcccagg agagcggcat ggatagacac cctgctgctt gcgccagcgccaggatcaac 1800 gtctagggcg cggactttat ttatttattt ctt 1833 <210> SEQ IDNO 81 <211> LENGTH: 39 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: A synthetic mutant ODCpeptide <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1)...(39)<223> OTHER INFORMATION: Xaa = any amino acid wherein one or more of theXaa residues are not the naturally occurring residue <400> SEQUENCE: 81His Gly Phe Xaa Xaa Xaa Met Xaa Xaa Gln Xaa Xaa Gly Thr Leu Pro 1 5 1015 Met Ser Cys Ala Gln Glu Ser Gly Xaa Xaa Arg His Pro Ala Ala Cys 20 2530 Ala Ser Ala Arg Ile Asn Val 35 <210> SEQ ID NO 82 <211> LENGTH: 13<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: A synthetic peptide <400> SEQUENCE: 82 Met Glu AspAla Lys Asn Ile Lys Lys Lys Ile Ala Val 1 5 10 <210> SEQ ID NO 83 <211>LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: A synthetic peptide <400> SEQUENCE: 83Met Gln Ile Phe Gly Gly His Pro Arg Asp Pro Val Thr Asp Ala Lys 1 5 1015 Asn Ile Lys Lys Lys Ile Ala Val 20 <210> SEQ ID NO 84 <211> LENGTH:20 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: A synthetic peptide <400> SEQUENCE: 84 Met GlnIle Phe Gly Gly His Val Thr Asp Ala Lys Asn Ile Lys Lys 1 5 10 15 LysIle Ala Val 20 <210> SEQ ID NO 85 <211> LENGTH: 24 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Asynthetic peptide <400> SEQUENCE: 85 Met Gln Ile Phe Gly Gly Glu Pro ArgAsp Pro Val Thr Asp Ala Lys 1 5 10 15 Asn Ile Lys Lys Lys Ile Ala Val 20<210> SEQ ID NO 86 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: A syntheticpeptide <400> SEQUENCE: 86 Met Gln Ile Phe Gly Gly Glu Val Thr Asp AlaLys Asn Ile Lys Lys 1 5 10 15 Lys Ile Ala Val 20 <210> SEQ ID NO 87<211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: A synthetic peptide <400>SEQUENCE: 87 Met Gln Ile Phe Gly Gly Tyr Pro Arg Asp Pro Val Thr Asp AlaLys 1 5 10 15 Asn Ile Lys Lys Lys Ile Ala Val 20 <210> SEQ ID NO 88<211> LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: A synthetic peptide <400>SEQUENCE: 88 Met Gln Ile Phe Gly Gly Tyr Pro Arg Asp Pro Glu Asp Ala LysAsn 1 5 10 15 Ile Lys Lys Lys Ile Ala Val 20

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleic acid sequence encoding a fusion polypeptide comprising areporter protein and at least two different heterologous proteindestabilization sequences, which fusion polypeptide has a reducedhalf-life relative to a corresponding reporter protein which lacks theheterologous protein destabilization sequences or has a reducedhalf-life relative to a corresponding reporter protein which has one ofthe heterologous protein destabilization sequences.
 2. An isolatednucleic acid molecule comprising a nucleic acid sequence comprising anopen reading frame for a reporter protein and at least two heterologousdestabilization sequences, wherein one of the heterologousdestabilization sequences is a mRNA destabilization sequence and anotheris a heterologous protein destabilization sequence.
 3. An isolatednucleic acid molecule comprising a nucleic acid sequence comprising anopen reading frame for a luciferase and at least one heterologousdestabilization sequence, wherein a majority of codons in the openreading frame for the luciferase are codons which are preferentiallyemployed in a selected host cell.
 4. The isolated nucleic acid moleculeof claim 1, 2 or 3 further comprising a promoter operably linked to thenucleic acid sequence.
 5. The isolated nucleic acid molecule of claim 4wherein the promoter is a regulatable promoter.
 6. The isolated nucleicacid molecule of claim 5 wherein the promoter is an inducible promoter.7. The isolated nucleic acid molecule of claim 5 wherein the promoter isa repressible promoter.
 8. The isolated nucleic acid molecule of claim 1further comprising a heterologous mRNA destabilization sequence.
 9. Theisolated nucleic acid molecule of claim 2 or 8 wherein the mRNAdestabilization is 3′ to the nucleic acid sequence.
 10. The isolatednucleic acid molecule of claim 1 or 2 wherein the nucleic acid sequenceencoding at least the reporter protein is optimized for expression in ahost cell.
 11. The isolated nucleic acid molecule of claim 1 or 2wherein the reporter protein encodes a luciferase.
 12. The isolatednucleic acid molecule of claim 1 wherein the reporter protein encodes abeetle luciferase.
 13. The isolated nucleic acid molecule of claim 12wherein the reporter protein encodes a click beetle luciferase.
 14. Theisolated nucleic acid molecule of claim 1 wherein the reporter proteinencodes an anthozoan luciferase protein.
 15. The isolated nucleic acidmolecule of claim 3 wherein the heterologous destabilization sequence isa protein destabilization sequence.
 16. The isolated nucleic acidmolecule of claim 3 wherein the heterologous destabilization sequence isa mRNA destabilization sequence.
 17. The isolated nucleic acid moleculeof claim 1, 2 or 3 wherein nucleic acid sequence comprises SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:70,SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75,SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, ora fragment thereof that encodes a fusion polypeptide with substantiallythe same activity as the corresponding full-length fusion polypeptideencoded by SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:66, SEQ ID NO:69, SEQID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79 or SEQ IDNO:80.
 18. The isolated nucleic acid molecule of claim 1 furthercomprising a mRNA destabilization sequence.
 19. The isolated molecule ofclaim 18 wherein one protein destabilization sequence is a PESTsequence.
 20. The isolated nucleic acid molecule of claim 1 or 2 whereinone heterologous protein destabilization sequence is a PEST sequence.21. The isolated nucleic acid molecule of claim 1 or 2 wherein oneheterologous protein destabilization sequence is from the C-terminus ofa mammalian ornithine decarboxylase.
 22. The isolated nucleic acidmolecule of claim 1 or 2 wherein one heterologous proteindestabilization sequence is a mutant omithine decarboxylase sequence.23. The isolated nucleic acid molecule of claim 21 wherein the mutantomithine decarboxylase sequence has an amino acid substitution at aposition corresponding to position 426, 427, 428, 430, 431, 433, 434,439 or 448 of murine omithine decarboxylase.
 24. The isolated nucleicacid molecule of claim 1 or 2 wherein one heterologous proteindestabilization sequence is CL1, CL2, CL6, CL9, CL10, CL11, CL12, CL15,CL16, CL17 or SL17.
 25. The isolated nucleic acid molecule of claim 1 or2 wherein one heterologous protein destabilization sequence is at theC-terminus of the reporter protein.
 26. The isolated nucleic acidmolecule of claim 1 or 2 wherein one heterologous proteindestabilization sequence at the N-terminus of the reporter protein. 27.The isolated nucleic acid molecule of claim 1 or 2 further comprising anubiquitin polypeptide at the N-terminus of the fusion polypeptide. 28.The isolated nucleic acid molecule of claim 27 wherein one of theheterologous protein destabilization sequences is at the C-terminus ofubiquitin.
 29. The isolated nucleic acid molecule of claim 28 whereinone of the heterologous protein destabilization sequences comprises aglutamnic acid or arginine residue.
 30. The isolated nucleic acidmolecule of claim 10 which encodes a fusion polypeptide with a half-lifeof expression of about 20 minutes.
 31. The isolated nucleic acidmolecule of claim 10 which encodes a fusion polypeptide with a half-lifeof expression of about 30 minutes.
 32. The isolated nucleic acidmolecule of claim 15 wherein the heterologous protein destabilizationsequence is a PEST sequence.
 33. The isolated nucleic acid molecule ofclaim 15 wherein the heterologous protein destabilization sequence isfrom the C-terminus of a mammalian omithine decarboxylase.
 34. Theisolated nucleic acid molecule of claim 15 wherein the heterologousprotein destabilization sequence is CL1, CL2, CL6, CL9, CL10, CL11,CL12, CL15, CL16, CL17 or SL17.
 35. A vector comprising the nucleic acidmolecule of claim 1, 2 or
 3. 36. The vector of claim 35 wherein thenucleic acid molecule is operably linked to a regulatable promoter. 37.The vector of claim 36 wherein the promoter is a repressible promoter.38. The vector of claim 34 wherein the nucleic acid molecule comprisesSEQ ID NO:49, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78,SEQ ID NO:79, SEQ ID NO:80 or a fragment thereof that encodes a fusionpolypeptide with substantially the same activity as the correspondingfull-length fusion polypeptide encoded by SEQ ID NO:49, SEQ ID NO:75,SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79 or SEQ ID NO:80.39. A fusion polypeptide encoded by the nucleic acid molecule of claim1, 2 or
 3. 40. The fusion polypeptide of claim 38 wherein the reporterprotein is chloramphenicol acetyltransferase, luciferase,beta-glucuronidase or beta-galactosidase.
 41. A host cell comprising thevector of claim
 35. 42. The host cell of claim 41 which is stablytransfected with the vector that encodes a fusion polypeptide comprisinga luminescent protein.
 43. The host cell of claim 42 wherein the signalemitted by the host cell comprising the vector is greater than thesignal emitted by a corresponding host cell comprising a vector whichlacks one or more of the destabilization sequences.
 44. A stable cellline comprising the vector of claim 35 wherein the signal emitted by thereporter protein is equal to or greater than a signal emitted by acorresponding stable cell line comprising a vector which lacks one ormore of the heterologous destabilization sequences.
 45. A method todetect a reporter protein in a cell, comprising: a) contacting a cellwith the vector of claim 35; and b) detecting or determining thepresence or amount of the reporter protein in the cell or a lysatethereof.